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<!DOCTYPE html>
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<title>The APOE isoforms differentially shape the transcriptomic and epigenomic landscapes of human microglia in a xenotransplantation model of Alzheimer’s disease</title>
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<h1 class="title">The APOE isoforms differentially shape the transcriptomic and epigenomic landscapes of human microglia in a xenotransplantation model of Alzheimer’s disease</h1>
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<p>This markdown contains all the code to generate the main and supplementary figures in:</p>
<p>Murphy, KB., Hu D., Wolfes, L., Mancuso, R., De Strooper, B., Marzi, SJ. The APOE isoforms differentially shape the transcriptomic and epigenomic landscapes of human microglia in a xenotransplantation model of Alzheimer’s disease.</p>
<div class="cell">
<details class="code-fold">
<summary>Code</summary>
<div class="sourceCode cell-code" id="cb1"><pre class="sourceCode r code-with-copy"><code class="sourceCode r"><span id="cb1-1"><a href="#cb1-1" aria-hidden="true" tabindex="-1"></a><span class="do">### Differential expression analysis using DeSeq2 </span><span class="al">###</span></span>
<span id="cb1-2"><a href="#cb1-2" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb1-3"><a href="#cb1-3" aria-hidden="true" tabindex="-1"></a><span class="co"># Define samples and groups </span></span>
<span id="cb1-4"><a href="#cb1-4" aria-hidden="true" tabindex="-1"></a>samples <span class="ot"><-</span> <span class="fu">c</span>(</span>
<span id="cb1-5"><a href="#cb1-5" aria-hidden="true" tabindex="-1"></a> <span class="fu">paste</span>(<span class="st">"E2-"</span>, <span class="dv">1</span><span class="sc">:</span><span class="dv">5</span>, <span class="at">sep=</span><span class="st">""</span>), </span>
<span id="cb1-6"><a href="#cb1-6" aria-hidden="true" tabindex="-1"></a> <span class="fu">paste</span>(<span class="st">"E3-"</span>, <span class="dv">2</span><span class="sc">:</span><span class="dv">5</span>, <span class="at">sep=</span><span class="st">""</span>), </span>
<span id="cb1-7"><a href="#cb1-7" aria-hidden="true" tabindex="-1"></a> <span class="fu">paste</span>(<span class="st">"E4-"</span>, <span class="dv">1</span><span class="sc">:</span><span class="dv">5</span>, <span class="at">sep=</span><span class="st">""</span>), </span>
<span id="cb1-8"><a href="#cb1-8" aria-hidden="true" tabindex="-1"></a> <span class="fu">paste</span>(<span class="st">"KO-"</span>, <span class="dv">1</span><span class="sc">:</span><span class="dv">5</span>, <span class="at">sep=</span><span class="st">""</span>)</span>
<span id="cb1-9"><a href="#cb1-9" aria-hidden="true" tabindex="-1"></a>)</span>
<span id="cb1-10"><a href="#cb1-10" aria-hidden="true" tabindex="-1"></a>group <span class="ot"><-</span> <span class="fu">c</span>(</span>
<span id="cb1-11"><a href="#cb1-11" aria-hidden="true" tabindex="-1"></a> <span class="fu">rep</span>(<span class="st">"E2"</span>, <span class="at">times =</span> <span class="dv">5</span>), </span>
<span id="cb1-12"><a href="#cb1-12" aria-hidden="true" tabindex="-1"></a> <span class="fu">rep</span>(<span class="st">"E3"</span>, <span class="at">times =</span> <span class="dv">4</span>), </span>
<span id="cb1-13"><a href="#cb1-13" aria-hidden="true" tabindex="-1"></a> <span class="fu">rep</span>(<span class="st">"E4"</span>, <span class="at">times =</span> <span class="dv">5</span>), </span>
<span id="cb1-14"><a href="#cb1-14" aria-hidden="true" tabindex="-1"></a> <span class="fu">rep</span>(<span class="st">"KO"</span>, <span class="at">times =</span> <span class="dv">5</span>)</span>
<span id="cb1-15"><a href="#cb1-15" aria-hidden="true" tabindex="-1"></a>)</span>
<span id="cb1-16"><a href="#cb1-16" aria-hidden="true" tabindex="-1"></a>colData <span class="ot"><-</span> <span class="fu">data.frame</span>(<span class="at">samples =</span> samples, <span class="at">group =</span> group)</span>
<span id="cb1-17"><a href="#cb1-17" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb1-18"><a href="#cb1-18" aria-hidden="true" tabindex="-1"></a><span class="do">## Hashed out code is to read in the Salmon quant.sf files and create a DeSeqDataset object ##</span></span>
<span id="cb1-19"><a href="#cb1-19" aria-hidden="true" tabindex="-1"></a><span class="co"># make tx2gene from gtf file </span></span>
<span id="cb1-20"><a href="#cb1-20" aria-hidden="true" tabindex="-1"></a><span class="co"># txdb <- TxDb.Hsapiens.UCSC.hg38.knownGene</span></span>
<span id="cb1-21"><a href="#cb1-21" aria-hidden="true" tabindex="-1"></a><span class="co"># key <- keys(txdb, keytype = "GENEID")</span></span>
<span id="cb1-22"><a href="#cb1-22" aria-hidden="true" tabindex="-1"></a><span class="co"># tx2gene <- select(txdb, keys = key, keytype = "GENEID", columns = "TXNAME")</span></span>
<span id="cb1-23"><a href="#cb1-23" aria-hidden="true" tabindex="-1"></a><span class="co"># tx2gene <- tx2gene[, c(2, 1)]</span></span>
<span id="cb1-24"><a href="#cb1-24" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb1-25"><a href="#cb1-25" aria-hidden="true" tabindex="-1"></a><span class="co"># import salmon quant files </span></span>
<span id="cb1-26"><a href="#cb1-26" aria-hidden="true" tabindex="-1"></a><span class="co"># files <- file.path(rna_dir, "salmon", samples, "quant.sf")</span></span>
<span id="cb1-27"><a href="#cb1-27" aria-hidden="true" tabindex="-1"></a><span class="co"># txi <- tximport(files, type = "salmon", tx2gene = tx2gene)</span></span>
<span id="cb1-28"><a href="#cb1-28" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb1-29"><a href="#cb1-29" aria-hidden="true" tabindex="-1"></a><span class="co"># make DeSeqDataset object </span></span>
<span id="cb1-30"><a href="#cb1-30" aria-hidden="true" tabindex="-1"></a><span class="co"># ddsTxi <- DESeqDataSetFromTximport(txi,</span></span>
<span id="cb1-31"><a href="#cb1-31" aria-hidden="true" tabindex="-1"></a><span class="co"># colData = colData,</span></span>
<span id="cb1-32"><a href="#cb1-32" aria-hidden="true" tabindex="-1"></a><span class="co"># design = ~ group) </span></span>
<span id="cb1-33"><a href="#cb1-33" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb1-34"><a href="#cb1-34" aria-hidden="true" tabindex="-1"></a><span class="co"># Read in DESeqDataset object </span></span>
<span id="cb1-35"><a href="#cb1-35" aria-hidden="true" tabindex="-1"></a>ddsTxi <span class="ot"><-</span> <span class="fu">readRDS</span>(<span class="st">"data/APOE_microglia_RNAseq_DESeqDataset_obj.rds"</span>)</span>
<span id="cb1-36"><a href="#cb1-36" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb1-37"><a href="#cb1-37" aria-hidden="true" tabindex="-1"></a><span class="co"># Remove samples with high APOE expression in the KO</span></span>
<span id="cb1-38"><a href="#cb1-38" aria-hidden="true" tabindex="-1"></a>indices_to_remove <span class="ot"><-</span> <span class="fu">which</span>(ddsTxi<span class="sc">$</span>samples <span class="sc">%in%</span> <span class="fu">c</span>(<span class="st">"KO-1"</span>, <span class="st">"KO-2"</span>))</span>
<span id="cb1-39"><a href="#cb1-39" aria-hidden="true" tabindex="-1"></a>ddsTxi_subset <span class="ot"><-</span> ddsTxi[, <span class="sc">-</span>indices_to_remove]</span>
<span id="cb1-40"><a href="#cb1-40" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb1-41"><a href="#cb1-41" aria-hidden="true" tabindex="-1"></a><span class="co"># Run differential expression analysis </span></span>
<span id="cb1-42"><a href="#cb1-42" aria-hidden="true" tabindex="-1"></a>dds <span class="ot"><-</span> <span class="fu">DESeq</span>(ddsTxi_subset) </span>
<span id="cb1-43"><a href="#cb1-43" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb1-44"><a href="#cb1-44" aria-hidden="true" tabindex="-1"></a><span class="co"># Run contrasts</span></span>
<span id="cb1-45"><a href="#cb1-45" aria-hidden="true" tabindex="-1"></a>contrasts <span class="ot"><-</span> <span class="fu">list</span>(</span>
<span id="cb1-46"><a href="#cb1-46" aria-hidden="true" tabindex="-1"></a> <span class="fu">c</span>(<span class="st">"group"</span>, <span class="st">"E2"</span>, <span class="st">"E3"</span>),</span>
<span id="cb1-47"><a href="#cb1-47" aria-hidden="true" tabindex="-1"></a> <span class="fu">c</span>(<span class="st">"group"</span>, <span class="st">"E4"</span>, <span class="st">"E3"</span>),</span>
<span id="cb1-48"><a href="#cb1-48" aria-hidden="true" tabindex="-1"></a> <span class="fu">c</span>(<span class="st">"group"</span>, <span class="st">"E4"</span>, <span class="st">"E2"</span>),</span>
<span id="cb1-49"><a href="#cb1-49" aria-hidden="true" tabindex="-1"></a> <span class="fu">c</span>(<span class="st">"group"</span>, <span class="st">"KO"</span>, <span class="st">"E3"</span>)</span>
<span id="cb1-50"><a href="#cb1-50" aria-hidden="true" tabindex="-1"></a> <span class="co"># Add more contrasts as needed</span></span>
<span id="cb1-51"><a href="#cb1-51" aria-hidden="true" tabindex="-1"></a>)</span>
<span id="cb1-52"><a href="#cb1-52" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb1-53"><a href="#cb1-53" aria-hidden="true" tabindex="-1"></a>res <span class="ot"><-</span> <span class="fu">lapply</span>(contrasts, <span class="cf">function</span>(con) <span class="fu">results</span>(dds, <span class="at">contrast =</span> con))</span>
<span id="cb1-54"><a href="#cb1-54" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb1-55"><a href="#cb1-55" aria-hidden="true" tabindex="-1"></a><span class="co"># Get gene symbols corresponding to entrez ids</span></span>
<span id="cb1-56"><a href="#cb1-56" aria-hidden="true" tabindex="-1"></a>ensembl <span class="ot"><-</span> <span class="fu">useEnsembl</span>(<span class="at">biomart =</span> <span class="st">"genes"</span>, <span class="at">dataset =</span> <span class="st">"hsapiens_gene_ensembl"</span>)</span>
<span id="cb1-57"><a href="#cb1-57" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb1-58"><a href="#cb1-58" aria-hidden="true" tabindex="-1"></a>convertEntrezToGeneSymbol <span class="ot"><-</span> <span class="cf">function</span>(res) {</span>
<span id="cb1-59"><a href="#cb1-59" aria-hidden="true" tabindex="-1"></a> res_df <span class="ot"><-</span> <span class="fu">as.data.frame</span>(res)</span>
<span id="cb1-60"><a href="#cb1-60" aria-hidden="true" tabindex="-1"></a> entrez_ids <span class="ot"><-</span> <span class="fu">rownames</span>(res_df)</span>
<span id="cb1-61"><a href="#cb1-61" aria-hidden="true" tabindex="-1"></a> </span>
<span id="cb1-62"><a href="#cb1-62" aria-hidden="true" tabindex="-1"></a> gene_symbols <span class="ot"><-</span> <span class="fu">getBM</span>(</span>
<span id="cb1-63"><a href="#cb1-63" aria-hidden="true" tabindex="-1"></a> <span class="at">attributes =</span> <span class="fu">c</span>(<span class="st">'hgnc_symbol'</span>, <span class="st">'entrezgene_id'</span>),</span>
<span id="cb1-64"><a href="#cb1-64" aria-hidden="true" tabindex="-1"></a> <span class="at">values =</span> entrez_ids,</span>
<span id="cb1-65"><a href="#cb1-65" aria-hidden="true" tabindex="-1"></a> <span class="at">mart =</span> ensembl</span>
<span id="cb1-66"><a href="#cb1-66" aria-hidden="true" tabindex="-1"></a> )</span>
<span id="cb1-67"><a href="#cb1-67" aria-hidden="true" tabindex="-1"></a> res_df_with_symbols <span class="ot"><-</span> <span class="fu">merge</span>(</span>
<span id="cb1-68"><a href="#cb1-68" aria-hidden="true" tabindex="-1"></a> gene_symbols, </span>
<span id="cb1-69"><a href="#cb1-69" aria-hidden="true" tabindex="-1"></a> res_df, </span>
<span id="cb1-70"><a href="#cb1-70" aria-hidden="true" tabindex="-1"></a> <span class="at">by.x =</span> <span class="st">"entrezgene_id"</span>, </span>
<span id="cb1-71"><a href="#cb1-71" aria-hidden="true" tabindex="-1"></a> <span class="at">by.y =</span> <span class="st">"row.names"</span></span>
<span id="cb1-72"><a href="#cb1-72" aria-hidden="true" tabindex="-1"></a> )</span>
<span id="cb1-73"><a href="#cb1-73" aria-hidden="true" tabindex="-1"></a> res_df_with_symbols <span class="ot"><-</span> res_df_with_symbols[<span class="fu">order</span>(res_df_with_symbols<span class="sc">$</span>padj), ]</span>
<span id="cb1-74"><a href="#cb1-74" aria-hidden="true" tabindex="-1"></a> <span class="fu">return</span>(res_df_with_symbols)</span>
<span id="cb1-75"><a href="#cb1-75" aria-hidden="true" tabindex="-1"></a>}</span>
<span id="cb1-76"><a href="#cb1-76" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb1-77"><a href="#cb1-77" aria-hidden="true" tabindex="-1"></a>res_annot <span class="ot"><-</span> <span class="fu">lapply</span>(res, convertEntrezToGeneSymbol)</span>
<span id="cb1-78"><a href="#cb1-78" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb1-79"><a href="#cb1-79" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb1-80"><a href="#cb1-80" aria-hidden="true" tabindex="-1"></a><span class="co"># Filter to remove genes with NA logFC and perform FDR correction</span></span>
<span id="cb1-81"><a href="#cb1-81" aria-hidden="true" tabindex="-1"></a>filter_adjust <span class="ot"><-</span> <span class="cf">function</span>(x) { </span>
<span id="cb1-82"><a href="#cb1-82" aria-hidden="true" tabindex="-1"></a> res_df <span class="ot"><-</span> x[<span class="fu">complete.cases</span>(x<span class="sc">$</span>log2FoldChange), ]</span>
<span id="cb1-83"><a href="#cb1-83" aria-hidden="true" tabindex="-1"></a> res_df<span class="sc">$</span>fdr <span class="ot"><-</span> <span class="fu">p.adjust</span>(res_df<span class="sc">$</span>pvalue, <span class="at">method =</span> <span class="st">"fdr"</span>)</span>
<span id="cb1-84"><a href="#cb1-84" aria-hidden="true" tabindex="-1"></a> res_df<span class="sc">$</span>padj <span class="ot"><-</span> <span class="cn">NULL</span></span>
<span id="cb1-85"><a href="#cb1-85" aria-hidden="true" tabindex="-1"></a> <span class="fu">return</span>(res_df)</span>
<span id="cb1-86"><a href="#cb1-86" aria-hidden="true" tabindex="-1"></a>}</span>
<span id="cb1-87"><a href="#cb1-87" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb1-88"><a href="#cb1-88" aria-hidden="true" tabindex="-1"></a>res_annot_filt <span class="ot"><-</span> <span class="fu">lapply</span>(res_annot, filter_adjust)</span>
<span id="cb1-89"><a href="#cb1-89" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb1-90"><a href="#cb1-90" aria-hidden="true" tabindex="-1"></a><span class="co"># Get results df for each contrast</span></span>
<span id="cb1-91"><a href="#cb1-91" aria-hidden="true" tabindex="-1"></a><span class="co"># Define a function to process each dataset</span></span>
<span id="cb1-92"><a href="#cb1-92" aria-hidden="true" tabindex="-1"></a>get_deg_res <span class="ot"><-</span> <span class="cf">function</span>(data) {</span>
<span id="cb1-93"><a href="#cb1-93" aria-hidden="true" tabindex="-1"></a> data <span class="sc">%>%</span></span>
<span id="cb1-94"><a href="#cb1-94" aria-hidden="true" tabindex="-1"></a> <span class="fu">na.omit</span>() <span class="sc">%>%</span></span>
<span id="cb1-95"><a href="#cb1-95" aria-hidden="true" tabindex="-1"></a> dplyr<span class="sc">::</span><span class="fu">distinct</span>(entrezgene_id, <span class="at">.keep_all =</span> <span class="cn">TRUE</span>) <span class="sc">%>%</span></span>
<span id="cb1-96"><a href="#cb1-96" aria-hidden="true" tabindex="-1"></a> <span class="fu">filter</span>(<span class="fu">complete.cases</span>(log2FoldChange))</span>
<span id="cb1-97"><a href="#cb1-97" aria-hidden="true" tabindex="-1"></a>}</span>
<span id="cb1-98"><a href="#cb1-98" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb1-99"><a href="#cb1-99" aria-hidden="true" tabindex="-1"></a><span class="co"># Apply the function to each dataset</span></span>
<span id="cb1-100"><a href="#cb1-100" aria-hidden="true" tabindex="-1"></a>E2vsE3_rna <span class="ot"><-</span> <span class="fu">get_deg_res</span>(res_annot_filt[[<span class="dv">1</span>]])</span>
<span id="cb1-101"><a href="#cb1-101" aria-hidden="true" tabindex="-1"></a>E4vsE3_rna <span class="ot"><-</span> <span class="fu">get_deg_res</span>(res_annot_filt[[<span class="dv">2</span>]])</span>
<span id="cb1-102"><a href="#cb1-102" aria-hidden="true" tabindex="-1"></a>E4vsE2_rna <span class="ot"><-</span> <span class="fu">get_deg_res</span>(res_annot_filt[[<span class="dv">3</span>]])</span>
<span id="cb1-103"><a href="#cb1-103" aria-hidden="true" tabindex="-1"></a>KOvsE3_rna <span class="ot"><-</span> <span class="fu">get_deg_res</span>(res_annot_filt[[<span class="dv">4</span>]])</span>
<span id="cb1-104"><a href="#cb1-104" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb1-105"><a href="#cb1-105" aria-hidden="true" tabindex="-1"></a>E2vsE3_rna_sig <span class="ot"><-</span> E2vsE3_rna[E2vsE3_rna<span class="sc">$</span>fdr<span class="sc"><</span><span class="fl">0.05</span>,]</span>
<span id="cb1-106"><a href="#cb1-106" aria-hidden="true" tabindex="-1"></a>E4vsE3_rna_sig <span class="ot"><-</span> E4vsE3_rna[E4vsE3_rna<span class="sc">$</span>fdr<span class="sc"><</span><span class="fl">0.05</span>,]</span>
<span id="cb1-107"><a href="#cb1-107" aria-hidden="true" tabindex="-1"></a>E4vsE2_rna_sig <span class="ot"><-</span> E4vsE2_rna[E4vsE2_rna<span class="sc">$</span>fdr<span class="sc"><</span><span class="fl">0.05</span>,]</span>
<span id="cb1-108"><a href="#cb1-108" aria-hidden="true" tabindex="-1"></a>KOvsE3_rna_sig <span class="ot"><-</span> KOvsE3_rna[KOvsE3_rna<span class="sc">$</span>fdr<span class="sc"><</span><span class="fl">0.05</span>,]</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
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<details class="code-fold">
<summary>Code</summary>
<div class="sourceCode cell-code" id="cb2"><pre class="sourceCode r code-with-copy"><code class="sourceCode r"><span id="cb2-1"><a href="#cb2-1" aria-hidden="true" tabindex="-1"></a><span class="do">### Differential chromatin accessibility analysis using DeSeq2 </span><span class="al">###</span></span>
<span id="cb2-2"><a href="#cb2-2" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb2-3"><a href="#cb2-3" aria-hidden="true" tabindex="-1"></a><span class="co"># Read in ATAC-seq counts</span></span>
<span id="cb2-4"><a href="#cb2-4" aria-hidden="true" tabindex="-1"></a><span class="co"># Peaks were called using MACS3 with a p-value cut off of 0.01</span></span>
<span id="cb2-5"><a href="#cb2-5" aria-hidden="true" tabindex="-1"></a>atac_counts <span class="ot"><-</span> <span class="fu">read.csv</span>(<span class="st">"data/APOE_microglia_ATACseq_counts.csv"</span>)</span>
<span id="cb2-6"><a href="#cb2-6" aria-hidden="true" tabindex="-1"></a><span class="fu">rownames</span>(atac_counts) <span class="ot"><-</span> atac_counts<span class="sc">$</span>Peak</span>
<span id="cb2-7"><a href="#cb2-7" aria-hidden="true" tabindex="-1"></a>atac_f_counts <span class="ot"><-</span> atac_counts[,<span class="sc">-</span><span class="fu">c</span>(<span class="dv">1</span><span class="sc">:</span><span class="dv">6</span>)]</span>
<span id="cb2-8"><a href="#cb2-8" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb2-9"><a href="#cb2-9" aria-hidden="true" tabindex="-1"></a><span class="co"># Remove KO samples with high APOE expression</span></span>
<span id="cb2-10"><a href="#cb2-10" aria-hidden="true" tabindex="-1"></a>atac_f2_counts <span class="ot"><-</span> atac_f_counts[, <span class="sc">!</span><span class="fu">names</span>(atac_f_counts) <span class="sc">%in%</span> <span class="fu">c</span>(<span class="st">"KO_1"</span>, <span class="st">"KO_2"</span>)]</span>
<span id="cb2-11"><a href="#cb2-11" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb2-12"><a href="#cb2-12" aria-hidden="true" tabindex="-1"></a><span class="co"># Get peaks</span></span>
<span id="cb2-13"><a href="#cb2-13" aria-hidden="true" tabindex="-1"></a>atac_peaks <span class="ot"><-</span> atac_counts[, <span class="fu">c</span>(<span class="st">"Chr"</span>, <span class="st">"Start"</span>, <span class="st">"End"</span>, <span class="st">"Peak"</span>)]</span>
<span id="cb2-14"><a href="#cb2-14" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb2-15"><a href="#cb2-15" aria-hidden="true" tabindex="-1"></a><span class="co"># Set up design matrix for differential accessibility analysis</span></span>
<span id="cb2-16"><a href="#cb2-16" aria-hidden="true" tabindex="-1"></a>group <span class="ot"><-</span> <span class="fu">factor</span>(<span class="fu">c</span>(<span class="fu">rep</span>(<span class="st">"E3"</span>, <span class="dv">5</span>), <span class="fu">rep</span>(<span class="st">"KO"</span>, <span class="dv">2</span>), <span class="fu">rep</span>(<span class="st">"E4"</span>, <span class="dv">4</span>), <span class="fu">rep</span>(<span class="st">"E2"</span>, <span class="dv">5</span>)))</span>
<span id="cb2-17"><a href="#cb2-17" aria-hidden="true" tabindex="-1"></a>design <span class="ot"><-</span> <span class="fu">model.matrix</span>(<span class="sc">~</span> <span class="dv">0</span> <span class="sc">+</span> group)</span>
<span id="cb2-18"><a href="#cb2-18" aria-hidden="true" tabindex="-1"></a><span class="fu">colnames</span>(design) <span class="ot"><-</span> <span class="fu">levels</span>(group)</span>
<span id="cb2-19"><a href="#cb2-19" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb2-20"><a href="#cb2-20" aria-hidden="true" tabindex="-1"></a><span class="co"># Keep peaks with counts that are meaningful for statistical analysis</span></span>
<span id="cb2-21"><a href="#cb2-21" aria-hidden="true" tabindex="-1"></a>keep <span class="ot"><-</span> <span class="fu">filterByExpr</span>(atac_f2_counts, design)</span>
<span id="cb2-22"><a href="#cb2-22" aria-hidden="true" tabindex="-1"></a>atac_f3_counts <span class="ot"><-</span> atac_f2_counts[keep,]</span>
<span id="cb2-23"><a href="#cb2-23" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb2-24"><a href="#cb2-24" aria-hidden="true" tabindex="-1"></a><span class="co"># DESeq2 analysis</span></span>
<span id="cb2-25"><a href="#cb2-25" aria-hidden="true" tabindex="-1"></a>meta <span class="ot"><-</span> <span class="fu">data.frame</span>(<span class="at">samples =</span> <span class="fu">colnames</span>(atac_f3_counts), group)</span>
<span id="cb2-26"><a href="#cb2-26" aria-hidden="true" tabindex="-1"></a>atac_dds <span class="ot"><-</span> <span class="fu">DESeqDataSetFromMatrix</span>(atac_f3_counts, meta, <span class="sc">~</span>group)</span>
<span id="cb2-27"><a href="#cb2-27" aria-hidden="true" tabindex="-1"></a>atac_dds <span class="ot"><-</span> <span class="fu">DESeq</span>(atac_dds)</span>
<span id="cb2-28"><a href="#cb2-28" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb2-29"><a href="#cb2-29" aria-hidden="true" tabindex="-1"></a><span class="co"># Function to generate differential accessibility results</span></span>
<span id="cb2-30"><a href="#cb2-30" aria-hidden="true" tabindex="-1"></a>get_dar_res <span class="ot"><-</span> <span class="cf">function</span>(contrast) {</span>
<span id="cb2-31"><a href="#cb2-31" aria-hidden="true" tabindex="-1"></a> dar <span class="ot"><-</span> <span class="fu">as.data.frame</span>(<span class="fu">results</span>(atac_dds, <span class="at">contrast =</span> contrast))</span>
<span id="cb2-32"><a href="#cb2-32" aria-hidden="true" tabindex="-1"></a> dar<span class="sc">$</span>Peak <span class="ot"><-</span> <span class="fu">row.names</span>(dar)</span>
<span id="cb2-33"><a href="#cb2-33" aria-hidden="true" tabindex="-1"></a> dar <span class="ot"><-</span> <span class="fu">merge</span>(atac_peaks, dar, <span class="at">by =</span> <span class="st">"Peak"</span>)</span>
<span id="cb2-34"><a href="#cb2-34" aria-hidden="true" tabindex="-1"></a> dar <span class="ot"><-</span> dar[<span class="fu">order</span>(dar<span class="sc">$</span>pvalue, <span class="at">decreasing =</span> <span class="cn">FALSE</span>), ]</span>
<span id="cb2-35"><a href="#cb2-35" aria-hidden="true" tabindex="-1"></a> dar<span class="sc">$</span>fdr <span class="ot"><-</span> <span class="fu">p.adjust</span>(dar<span class="sc">$</span>pvalue, <span class="at">method =</span> <span class="st">"fdr"</span>)</span>
<span id="cb2-36"><a href="#cb2-36" aria-hidden="true" tabindex="-1"></a> dar<span class="sc">$</span>diffAccessible <span class="ot"><-</span> <span class="st">"Not sig"</span></span>
<span id="cb2-37"><a href="#cb2-37" aria-hidden="true" tabindex="-1"></a> dar<span class="sc">$</span>diffAccessible[dar<span class="sc">$</span>log2FoldChange <span class="sc">></span> <span class="dv">0</span> <span class="sc">&</span> dar<span class="sc">$</span>fdr <span class="sc"><</span> <span class="fl">0.05</span>] <span class="ot"><-</span> <span class="st">"Up"</span></span>
<span id="cb2-38"><a href="#cb2-38" aria-hidden="true" tabindex="-1"></a> dar<span class="sc">$</span>diffAccessible[dar<span class="sc">$</span>log2FoldChange <span class="sc"><</span> <span class="dv">0</span> <span class="sc">&</span> dar<span class="sc">$</span>fdr <span class="sc"><</span> <span class="fl">0.05</span>] <span class="ot"><-</span> <span class="st">"Down"</span></span>
<span id="cb2-39"><a href="#cb2-39" aria-hidden="true" tabindex="-1"></a> <span class="fu">return</span>(dar)</span>
<span id="cb2-40"><a href="#cb2-40" aria-hidden="true" tabindex="-1"></a>}</span>
<span id="cb2-41"><a href="#cb2-41" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb2-42"><a href="#cb2-42" aria-hidden="true" tabindex="-1"></a><span class="co"># Generate DAR results for each contrast</span></span>
<span id="cb2-43"><a href="#cb2-43" aria-hidden="true" tabindex="-1"></a>E2vsE3_dar <span class="ot"><-</span> <span class="fu">get_dar_res</span>(<span class="fu">c</span>(<span class="st">"group"</span>, <span class="st">"E2"</span>, <span class="st">"E3"</span>))</span>
<span id="cb2-44"><a href="#cb2-44" aria-hidden="true" tabindex="-1"></a>E4vsE2_dar <span class="ot"><-</span> <span class="fu">get_dar_res</span>(<span class="fu">c</span>(<span class="st">"group"</span>, <span class="st">"E4"</span>, <span class="st">"E2"</span>))</span>
<span id="cb2-45"><a href="#cb2-45" aria-hidden="true" tabindex="-1"></a>E4vsE3_dar <span class="ot"><-</span> <span class="fu">get_dar_res</span>(<span class="fu">c</span>(<span class="st">"group"</span>, <span class="st">"E4"</span>, <span class="st">"E3"</span>))</span>
<span id="cb2-46"><a href="#cb2-46" aria-hidden="true" tabindex="-1"></a>KOvsE3_dar <span class="ot"><-</span> <span class="fu">get_dar_res</span>(<span class="fu">c</span>(<span class="st">"group"</span>, <span class="st">"KO"</span>, <span class="st">"E3"</span>))</span>
<span id="cb2-47"><a href="#cb2-47" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb2-48"><a href="#cb2-48" aria-hidden="true" tabindex="-1"></a><span class="co"># Annotate peaks</span></span>
<span id="cb2-49"><a href="#cb2-49" aria-hidden="true" tabindex="-1"></a>txdb <span class="ot"><-</span> TxDb.Hsapiens.UCSC.hg38.knownGene</span>
<span id="cb2-50"><a href="#cb2-50" aria-hidden="true" tabindex="-1"></a>peaks_annot <span class="ot"><-</span> <span class="fu">suppressMessages</span>(<span class="fu">as.data.frame</span>(<span class="fu">annotatePeak</span>(</span>
<span id="cb2-51"><a href="#cb2-51" aria-hidden="true" tabindex="-1"></a> <span class="fu">makeGRangesFromDataFrame</span>(atac_peaks[, <span class="dv">1</span><span class="sc">:</span><span class="dv">3</span>]), </span>
<span id="cb2-52"><a href="#cb2-52" aria-hidden="true" tabindex="-1"></a> <span class="at">TxDb =</span> txdb, </span>
<span id="cb2-53"><a href="#cb2-53" aria-hidden="true" tabindex="-1"></a> <span class="at">tssRegion =</span> <span class="fu">c</span>(<span class="sc">-</span><span class="dv">3000</span>, <span class="dv">3000</span>), </span>
<span id="cb2-54"><a href="#cb2-54" aria-hidden="true" tabindex="-1"></a> <span class="at">annoDb =</span> <span class="st">"org.Hs.eg.db"</span></span>
<span id="cb2-55"><a href="#cb2-55" aria-hidden="true" tabindex="-1"></a>)))</span>
<span id="cb2-56"><a href="#cb2-56" aria-hidden="true" tabindex="-1"></a>peaks_annot<span class="sc">$</span>peak <span class="ot"><-</span> <span class="fu">row.names</span>(atac_peaks)</span>
<span id="cb2-57"><a href="#cb2-57" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb2-58"><a href="#cb2-58" aria-hidden="true" tabindex="-1"></a><span class="co"># Function to merge DAR with annotation and add contrast information</span></span>
<span id="cb2-59"><a href="#cb2-59" aria-hidden="true" tabindex="-1"></a>annotate_dar <span class="ot"><-</span> <span class="cf">function</span>(dar, contrast_name) {</span>
<span id="cb2-60"><a href="#cb2-60" aria-hidden="true" tabindex="-1"></a> dar_annot <span class="ot"><-</span> <span class="fu">merge</span>(</span>
<span id="cb2-61"><a href="#cb2-61" aria-hidden="true" tabindex="-1"></a> dar, </span>
<span id="cb2-62"><a href="#cb2-62" aria-hidden="true" tabindex="-1"></a> peaks_annot, </span>
<span id="cb2-63"><a href="#cb2-63" aria-hidden="true" tabindex="-1"></a> <span class="at">by.x =</span> <span class="fu">c</span>(<span class="st">"Chr"</span>, <span class="st">"Start"</span>, <span class="st">"End"</span>, <span class="st">"Peak"</span>), </span>
<span id="cb2-64"><a href="#cb2-64" aria-hidden="true" tabindex="-1"></a> <span class="at">by.y =</span> <span class="fu">c</span>(<span class="st">"seqnames"</span>, <span class="st">"start"</span>, <span class="st">"end"</span>, <span class="st">"peak"</span>)</span>
<span id="cb2-65"><a href="#cb2-65" aria-hidden="true" tabindex="-1"></a> )</span>
<span id="cb2-66"><a href="#cb2-66" aria-hidden="true" tabindex="-1"></a> dar_annot<span class="sc">$</span>contrast <span class="ot"><-</span> contrast_name</span>
<span id="cb2-67"><a href="#cb2-67" aria-hidden="true" tabindex="-1"></a> dar_annot <span class="ot"><-</span> dar_annot[<span class="fu">order</span>(dar_annot<span class="sc">$</span>pvalue, <span class="at">decreasing =</span> <span class="cn">FALSE</span>), ]</span>
<span id="cb2-68"><a href="#cb2-68" aria-hidden="true" tabindex="-1"></a> <span class="fu">return</span>(dar_annot)</span>
<span id="cb2-69"><a href="#cb2-69" aria-hidden="true" tabindex="-1"></a>}</span>
<span id="cb2-70"><a href="#cb2-70" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb2-71"><a href="#cb2-71" aria-hidden="true" tabindex="-1"></a><span class="co"># Annotate DAR results for each contrast</span></span>
<span id="cb2-72"><a href="#cb2-72" aria-hidden="true" tabindex="-1"></a>E2vsE3_atac_annot <span class="ot"><-</span> <span class="fu">annotate_dar</span>(E2vsE3_dar, <span class="st">"E2vsE3"</span>)</span>
<span id="cb2-73"><a href="#cb2-73" aria-hidden="true" tabindex="-1"></a>E4vsE3_atac_annot <span class="ot"><-</span> <span class="fu">annotate_dar</span>(E4vsE3_dar, <span class="st">"E4vsE3"</span>)</span>
<span id="cb2-74"><a href="#cb2-74" aria-hidden="true" tabindex="-1"></a>E4vsE2_atac_annot <span class="ot"><-</span> <span class="fu">annotate_dar</span>(E4vsE2_dar, <span class="st">"E4vsE2"</span>)</span>
<span id="cb2-75"><a href="#cb2-75" aria-hidden="true" tabindex="-1"></a>KOvsE3_atac_annot <span class="ot"><-</span> <span class="fu">annotate_dar</span>(KOvsE3_dar, <span class="st">"KOvsE3"</span>)</span>
<span id="cb2-76"><a href="#cb2-76" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb2-77"><a href="#cb2-77" aria-hidden="true" tabindex="-1"></a><span class="co"># Filter significant DAR results</span></span>
<span id="cb2-78"><a href="#cb2-78" aria-hidden="true" tabindex="-1"></a>E2vsE3_atac_sig <span class="ot"><-</span> E2vsE3_atac_annot[E2vsE3_atac_annot<span class="sc">$</span>fdr <span class="sc"><</span> <span class="fl">0.05</span>,]</span>
<span id="cb2-79"><a href="#cb2-79" aria-hidden="true" tabindex="-1"></a>E4vsE3_atac_sig <span class="ot"><-</span> E4vsE3_atac_annot[E4vsE3_atac_annot<span class="sc">$</span>fdr <span class="sc"><</span> <span class="fl">0.05</span>,]</span>
<span id="cb2-80"><a href="#cb2-80" aria-hidden="true" tabindex="-1"></a>E4vsE2_atac_sig <span class="ot"><-</span> E4vsE2_atac_annot[E4vsE2_atac_annot<span class="sc">$</span>fdr <span class="sc"><</span> <span class="fl">0.05</span>,]</span>
<span id="cb2-81"><a href="#cb2-81" aria-hidden="true" tabindex="-1"></a>KOvsE3_atac_sig <span class="ot"><-</span> KOvsE3_atac_annot[KOvsE3_atac_annot<span class="sc">$</span>fdr <span class="sc"><</span> <span class="fl">0.05</span>,]</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
</details>
</div>
<p><strong>Figure 1: Transcriptomic and epigenomic profiling of xenotransplanted microglia reveals changes to their regulation in Alzheimer’s disease across the different <em>APOE</em> isoforms.</strong></p>
<p><img src="figures/chapter5_workflow.png" class="img-fluid"></p>
<p><strong>Figure 1a) Experimental design for xenotransplantation of iPSC-derived human microglia into the brains of AppNL-G-F mice (<em>APOE2</em> = 5, <em>APOE3</em> = 5, E4 = 4, <em>APOE</em>-KO = 2) and high-quality transcriptomic data across 17 mice (<em>APOE2</em> = 5, <em>APOE4</em> = 4, <em>APOE4</em> = 5, <em>APOE</em>-KO = 3), followed by transcriptomic and chromatin accessibility profiling at 12 months.</strong></p>
<div class="cell" data-layout-align="center">
<details class="code-fold">
<summary>Code</summary>
<div class="sourceCode cell-code" id="cb3"><pre class="sourceCode r code-with-copy"><code class="sourceCode r"><span id="cb3-1"><a href="#cb3-1" aria-hidden="true" tabindex="-1"></a><span class="do">## Figure 1b: apoe expression ##</span></span>
<span id="cb3-2"><a href="#cb3-2" aria-hidden="true" tabindex="-1"></a>apoe <span class="ot"><-</span> <span class="fu">plotCounts</span>(dds, <span class="at">gene=</span><span class="st">"348"</span>, <span class="at">intgroup=</span><span class="st">"group"</span>, <span class="at">returnData =</span> <span class="cn">TRUE</span>)</span>
<span id="cb3-3"><a href="#cb3-3" aria-hidden="true" tabindex="-1"></a>my_comparisons <span class="ot"><-</span> <span class="fu">list</span>(<span class="fu">c</span>(<span class="st">"E4"</span>, <span class="st">"KO"</span>), <span class="fu">c</span>(<span class="st">"E3"</span>, <span class="st">"KO"</span>), <span class="fu">c</span>(<span class="st">"E2"</span>,<span class="st">"KO"</span>), <span class="fu">c</span>(<span class="st">"E4"</span>, <span class="st">"E3"</span>))</span>
<span id="cb3-4"><a href="#cb3-4" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb3-5"><a href="#cb3-5" aria-hidden="true" tabindex="-1"></a>apoe_exp <span class="ot"><-</span> <span class="fu">ggplot</span>(apoe, <span class="fu">aes</span>(<span class="at">x=</span>group, <span class="at">y=</span>count, <span class="at">fill=</span>group)) <span class="sc">+</span> </span>
<span id="cb3-6"><a href="#cb3-6" aria-hidden="true" tabindex="-1"></a> <span class="fu">geom_boxplot</span>() <span class="sc">+</span> </span>
<span id="cb3-7"><a href="#cb3-7" aria-hidden="true" tabindex="-1"></a> <span class="fu">theme_classic</span>() <span class="sc">+</span> </span>
<span id="cb3-8"><a href="#cb3-8" aria-hidden="true" tabindex="-1"></a> <span class="fu">scale_fill_manual</span>(<span class="at">values =</span> <span class="fu">ghibli_palette</span>(<span class="st">"YesterdayMedium"</span>, <span class="at">direction =</span> <span class="sc">-</span><span class="dv">1</span>)) <span class="sc">+</span> </span>
<span id="cb3-9"><a href="#cb3-9" aria-hidden="true" tabindex="-1"></a> <span class="fu">ylab</span>(<span class="st">"Counts"</span>) <span class="sc">+</span> </span>
<span id="cb3-10"><a href="#cb3-10" aria-hidden="true" tabindex="-1"></a> <span class="fu">ggtitle</span>(<span class="st">"APOE"</span>) <span class="sc">+</span> </span>
<span id="cb3-11"><a href="#cb3-11" aria-hidden="true" tabindex="-1"></a> <span class="fu">xlab</span>(<span class="st">""</span>) <span class="sc">+</span> </span>
<span id="cb3-12"><a href="#cb3-12" aria-hidden="true" tabindex="-1"></a> <span class="fu">theme</span>(<span class="at">text =</span> <span class="fu">element_text</span>(<span class="at">size =</span> <span class="dv">20</span>), <span class="at">plot.title =</span> <span class="fu">element_text</span>(<span class="at">hjust =</span> <span class="fl">0.5</span>, <span class="at">face=</span><span class="fu">c</span>(<span class="st">"italic"</span>))) <span class="sc">+</span></span>
<span id="cb3-13"><a href="#cb3-13" aria-hidden="true" tabindex="-1"></a> <span class="fu">stat_compare_means</span>(<span class="at">comparisons =</span> my_comparisons, <span class="at">method=</span><span class="st">"t.test"</span>, <span class="at">size=</span><span class="dv">4</span>) <span class="sc">+</span> </span>
<span id="cb3-14"><a href="#cb3-14" aria-hidden="true" tabindex="-1"></a> <span class="fu">geom_jitter</span>() <span class="sc">+</span></span>
<span id="cb3-15"><a href="#cb3-15" aria-hidden="true" tabindex="-1"></a> <span class="fu">labs</span>(<span class="at">fill=</span><span class="cn">NULL</span>)</span>
<span id="cb3-16"><a href="#cb3-16" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb3-17"><a href="#cb3-17" aria-hidden="true" tabindex="-1"></a><span class="fu">plot</span>(apoe_exp)</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
</details>
<div class="cell-output-display">
<div class="quarto-figure quarto-figure-center">
<figure class="figure">
<p><img src="manuscript_figures_files/figure-html/figure_1b-1.png" class="img-fluid quarto-figure quarto-figure-center figure-img" width="480"></p>
</figure>
</div>
</div>
</div>
<p><strong>Figure 1b) Boxplot of expression profiles of <em>APOE</em>, confirming the knockout.</strong></p>
<div class="cell" data-layout-align="center">
<details class="code-fold">
<summary>Code</summary>
<div class="sourceCode cell-code" id="cb4"><pre class="sourceCode r code-with-copy"><code class="sourceCode r"><span id="cb4-1"><a href="#cb4-1" aria-hidden="true" tabindex="-1"></a><span class="do">## Figure 1c: chromatin accessibility around APOE locus ##</span></span>
<span id="cb4-2"><a href="#cb4-2" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb4-3"><a href="#cb4-3" aria-hidden="true" tabindex="-1"></a><span class="co"># Get TSS of APOE and plot datatrack </span></span>
<span id="cb4-4"><a href="#cb4-4" aria-hidden="true" tabindex="-1"></a>hg38_genes <span class="ot"><-</span> <span class="fu">suppressMessages</span>(<span class="fu">genes</span>(TxDb.Hsapiens.UCSC.hg38.knownGene))</span>
<span id="cb4-5"><a href="#cb4-5" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb4-6"><a href="#cb4-6" aria-hidden="true" tabindex="-1"></a>start <span class="ot"><-</span> <span class="fu">start</span>(hg38_genes[hg38_genes<span class="sc">$</span>gene_id <span class="sc">%in%</span> <span class="st">"348"</span>,]) <span class="sc">-</span> <span class="dv">2000</span> </span>
<span id="cb4-7"><a href="#cb4-7" aria-hidden="true" tabindex="-1"></a>end <span class="ot"><-</span> <span class="fu">end</span>(hg38_genes[hg38_genes<span class="sc">$</span>gene_id <span class="sc">%in%</span> <span class="st">"348"</span>,]) <span class="sc">+</span> <span class="dv">2000</span></span>
<span id="cb4-8"><a href="#cb4-8" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb4-9"><a href="#cb4-9" aria-hidden="true" tabindex="-1"></a><span class="co"># Plot data tracks </span></span>
<span id="cb4-10"><a href="#cb4-10" aria-hidden="true" tabindex="-1"></a>track_list <span class="ot"><-</span> <span class="fu">c</span>(<span class="at">E3 =</span> <span class="st">"data/E3-1.bw"</span>, <span class="at">E4 =</span> <span class="st">"data/E4-1.bw"</span>, <span class="at">E2 =</span> <span class="st">"data/E2-1.bw"</span>, <span class="at">KO =</span> <span class="st">"data/KO-1.bw"</span>)</span>
<span id="cb4-11"><a href="#cb4-11" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb4-12"><a href="#cb4-12" aria-hidden="true" tabindex="-1"></a><span class="co"># Set apoe track region</span></span>
<span id="cb4-13"><a href="#cb4-13" aria-hidden="true" tabindex="-1"></a>apoe_region <span class="ot"><-</span> <span class="fu">suppressMessages</span>(<span class="fu">toGRanges</span>(<span class="fu">data.frame</span>(<span class="at">chr=</span><span class="st">"chr19"</span>, <span class="at">start=</span>start, <span class="at">end=</span>end)))</span>
<span id="cb4-14"><a href="#cb4-14" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb4-15"><a href="#cb4-15" aria-hidden="true" tabindex="-1"></a><span class="co"># Set plotting parameters</span></span>
<span id="cb4-16"><a href="#cb4-16" aria-hidden="true" tabindex="-1"></a>pp <span class="ot"><-</span> <span class="fu">getDefaultPlotParams</span>(<span class="at">plot.type=</span><span class="dv">1</span>)</span>
<span id="cb4-17"><a href="#cb4-17" aria-hidden="true" tabindex="-1"></a>pp<span class="sc">$</span>leftmargin <span class="ot"><-</span> <span class="fl">0.15</span></span>
<span id="cb4-18"><a href="#cb4-18" aria-hidden="true" tabindex="-1"></a>pp<span class="sc">$</span>topmargin <span class="ot"><-</span> <span class="dv">15</span></span>
<span id="cb4-19"><a href="#cb4-19" aria-hidden="true" tabindex="-1"></a>pp<span class="sc">$</span>bottommargin <span class="ot"><-</span> <span class="dv">15</span></span>
<span id="cb4-20"><a href="#cb4-20" aria-hidden="true" tabindex="-1"></a>pp<span class="sc">$</span>ideogramheight <span class="ot"><-</span> <span class="dv">5</span></span>
<span id="cb4-21"><a href="#cb4-21" aria-hidden="true" tabindex="-1"></a>pp<span class="sc">$</span>data1inmargin <span class="ot"><-</span> <span class="dv">10</span></span>
<span id="cb4-22"><a href="#cb4-22" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb4-23"><a href="#cb4-23" aria-hidden="true" tabindex="-1"></a>kp <span class="ot"><-</span> <span class="fu">plotKaryotype</span>(<span class="at">zoom =</span> apoe_region, <span class="at">cex=</span><span class="fl">1.7</span>, <span class="at">plot.params =</span> pp)</span>
<span id="cb4-24"><a href="#cb4-24" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb4-25"><a href="#cb4-25" aria-hidden="true" tabindex="-1"></a><span class="fu">kpAddBaseNumbers</span>(kp, <span class="at">tick.dist =</span> <span class="dv">3000</span>, <span class="at">minor.tick.dist =</span> <span class="dv">1500</span>, <span class="at">add.units =</span> <span class="cn">TRUE</span>, <span class="at">cex=</span><span class="fl">1.5</span>, <span class="at">digits =</span> <span class="dv">6</span>)</span>
<span id="cb4-26"><a href="#cb4-26" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb4-27"><a href="#cb4-27" aria-hidden="true" tabindex="-1"></a>apoe_data <span class="ot"><-</span> <span class="fu">suppressMessages</span>(<span class="fu">makeGenesDataFromTxDb</span>(TxDb.Hsapiens.UCSC.hg38.knownGene, <span class="at">karyoplot=</span>kp, <span class="at">plot.transcripts =</span> <span class="cn">TRUE</span>, <span class="at">plot.transcripts.structure =</span> <span class="cn">TRUE</span>))</span>
<span id="cb4-28"><a href="#cb4-28" aria-hidden="true" tabindex="-1"></a>apoe_data<span class="sc">$</span>genes<span class="sc">$</span>name <span class="ot"><-</span> <span class="st">"APOE"</span></span>
<span id="cb4-29"><a href="#cb4-29" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb4-30"><a href="#cb4-30" aria-hidden="true" tabindex="-1"></a><span class="co"># Plotting tracks</span></span>
<span id="cb4-31"><a href="#cb4-31" aria-hidden="true" tabindex="-1"></a>plot_track <span class="ot"><-</span> <span class="cf">function</span>(track_name, track_position, track_colour, r1_value) {</span>
<span id="cb4-32"><a href="#cb4-32" aria-hidden="true" tabindex="-1"></a> bigwig.file <span class="ot"><-</span> track_list[[track_name]]</span>
<span id="cb4-33"><a href="#cb4-33" aria-hidden="true" tabindex="-1"></a> track_margin <span class="ot"><-</span> <span class="fl">0.1</span></span>
<span id="cb4-34"><a href="#cb4-34" aria-hidden="true" tabindex="-1"></a> genes_track_at <span class="ot"><-</span> <span class="fu">autotrack</span>(<span class="dv">1</span>, <span class="dv">1</span>, <span class="at">r0=</span><span class="dv">0</span>, <span class="at">r1=</span>track_margin)</span>
<span id="cb4-35"><a href="#cb4-35" aria-hidden="true" tabindex="-1"></a> at <span class="ot"><-</span> <span class="fu">autotrack</span>(track_position, <span class="fu">length</span>(track_list), <span class="at">r0=</span>genes_track_at<span class="sc">$</span>r1 <span class="sc">+</span> track_margin, <span class="at">margin =</span> <span class="fl">0.2</span>)</span>
<span id="cb4-36"><a href="#cb4-36" aria-hidden="true" tabindex="-1"></a> kp <span class="ot"><-</span> <span class="fu">kpPlotGenes</span>(kp, <span class="at">data=</span>apoe_data, <span class="at">r0=</span>genes_track_at<span class="sc">$</span>r0, <span class="at">r1=</span>genes_track_at<span class="sc">$</span>r1, <span class="at">gene.name.cex=</span><span class="dv">2</span>)</span>
<span id="cb4-37"><a href="#cb4-37" aria-hidden="true" tabindex="-1"></a> kp <span class="ot"><-</span> <span class="fu">kpPlotBigWig</span>(kp, <span class="at">data=</span>bigwig.file, <span class="at">ymax=</span><span class="st">"visible.region"</span>, <span class="at">r0=</span>at<span class="sc">$</span>r0, <span class="at">r1=</span>r1_value, <span class="at">col =</span> track_colour)</span>
<span id="cb4-38"><a href="#cb4-38" aria-hidden="true" tabindex="-1"></a> <span class="fu">kpAxis</span>(kp, <span class="at">ymin=</span><span class="dv">0</span>, <span class="at">ymax=</span><span class="dv">116</span>, <span class="at">numticks =</span> <span class="dv">2</span>, <span class="at">r0=</span>at<span class="sc">$</span>r0, <span class="at">r1=</span>at<span class="sc">$</span>r1, <span class="at">cex =</span> <span class="fl">1.5</span>)</span>
<span id="cb4-39"><a href="#cb4-39" aria-hidden="true" tabindex="-1"></a> <span class="fu">kpAddLabels</span>(kp, <span class="at">labels =</span> track_name, <span class="at">r0=</span>at<span class="sc">$</span>r0, <span class="at">r1=</span>at<span class="sc">$</span>r1, <span class="at">cex=</span><span class="dv">2</span>, <span class="at">label.margin =</span> <span class="fl">0.035</span>, <span class="at">col =</span> track_colour)</span>
<span id="cb4-40"><a href="#cb4-40" aria-hidden="true" tabindex="-1"></a>}</span>
<span id="cb4-41"><a href="#cb4-41" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb4-42"><a href="#cb4-42" aria-hidden="true" tabindex="-1"></a><span class="fu">plot_track</span>(<span class="st">"KO"</span>, <span class="dv">1</span>, <span class="st">"#4D6D93FF"</span>, <span class="fl">0.26745</span>)</span>
<span id="cb4-43"><a href="#cb4-43" aria-hidden="true" tabindex="-1"></a><span class="fu">plot_track</span>(<span class="st">"E4"</span>, <span class="dv">2</span>, <span class="st">"#6FB382FF"</span>, <span class="fl">0.52827</span>)</span>
<span id="cb4-44"><a href="#cb4-44" aria-hidden="true" tabindex="-1"></a><span class="fu">plot_track</span>(<span class="st">"E3"</span>, <span class="dv">3</span>, <span class="st">"#DCCA2CFF"</span>, <span class="fl">0.73274</span>)</span>
<span id="cb4-45"><a href="#cb4-45" aria-hidden="true" tabindex="-1"></a><span class="fu">plot_track</span>(<span class="st">"E2"</span>, <span class="dv">4</span>, <span class="st">"#92BBD9FF"</span>, <span class="dv">1</span>)</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
</details>
<div class="cell-output-display">
<div class="quarto-figure quarto-figure-center">
<figure class="figure">
<p><img src="manuscript_figures_files/figure-html/figure_1c-1.png" class="img-fluid quarto-figure quarto-figure-center figure-img" width="576"></p>
</figure>
</div>
</div>
</div>
<p><strong>Figure 1c) Genome tracks showing chromatin accessibility signals of all APOE groups around the <em>APOE</em> locus.</strong></p>
<div class="cell" data-layout-align="center">
<details class="code-fold">
<summary>Code</summary>
<div class="sourceCode cell-code" id="cb5"><pre class="sourceCode r code-with-copy"><code class="sourceCode r"><span id="cb5-1"><a href="#cb5-1" aria-hidden="true" tabindex="-1"></a><span class="do">## Figure 1d: no of DEGs ##</span></span>
<span id="cb5-2"><a href="#cb5-2" aria-hidden="true" tabindex="-1"></a>KOvsE3_rna<span class="sc">$</span>comparison <span class="ot"><-</span> <span class="st">"KOvsE3"</span></span>
<span id="cb5-3"><a href="#cb5-3" aria-hidden="true" tabindex="-1"></a>E4vsE2_rna<span class="sc">$</span>comparison <span class="ot"><-</span> <span class="st">"E4vsE2"</span></span>
<span id="cb5-4"><a href="#cb5-4" aria-hidden="true" tabindex="-1"></a>E4vsE3_rna<span class="sc">$</span>comparison <span class="ot"><-</span> <span class="st">"E4vsE3"</span></span>
<span id="cb5-5"><a href="#cb5-5" aria-hidden="true" tabindex="-1"></a>E2vsE3_rna<span class="sc">$</span>comparison <span class="ot"><-</span> <span class="st">"E2vsE3"</span></span>
<span id="cb5-6"><a href="#cb5-6" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb5-7"><a href="#cb5-7" aria-hidden="true" tabindex="-1"></a>comparisons <span class="ot"><-</span> <span class="fu">rbind</span>(E2vsE3_rna, E4vsE3_rna, E4vsE2_rna, KOvsE3_rna)</span>
<span id="cb5-8"><a href="#cb5-8" aria-hidden="true" tabindex="-1"></a>comparisons<span class="sc">$</span>diffExpressed <span class="ot"><-</span> <span class="st">"Not sig"</span></span>
<span id="cb5-9"><a href="#cb5-9" aria-hidden="true" tabindex="-1"></a>comparisons<span class="sc">$</span>diffExpressed[comparisons<span class="sc">$</span>log2FoldChange <span class="sc">></span> <span class="dv">0</span> <span class="sc">&</span> comparisons<span class="sc">$</span>fdr <span class="sc"><</span> <span class="fl">0.05</span>] <span class="ot"><-</span> <span class="st">"Up"</span></span>
<span id="cb5-10"><a href="#cb5-10" aria-hidden="true" tabindex="-1"></a>comparisons<span class="sc">$</span>diffExpressed[comparisons<span class="sc">$</span>log2FoldChange <span class="sc"><</span> <span class="dv">0</span> <span class="sc">&</span> comparisons<span class="sc">$</span>fdr <span class="sc"><</span> <span class="fl">0.05</span>] <span class="ot"><-</span> <span class="st">"Down"</span></span>
<span id="cb5-11"><a href="#cb5-11" aria-hidden="true" tabindex="-1"></a>comparisons<span class="sc">$</span>diffExpressed <span class="ot"><-</span> <span class="fu">factor</span>(comparisons<span class="sc">$</span>diffExpressed, <span class="at">levels=</span><span class="fu">c</span>(<span class="st">"Up"</span>, <span class="st">"Down"</span>, <span class="st">"Not sig"</span>))</span>
<span id="cb5-12"><a href="#cb5-12" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb5-13"><a href="#cb5-13" aria-hidden="true" tabindex="-1"></a>degs_plot <span class="ot"><-</span> <span class="fu">ggplot</span>(comparisons[<span class="sc">!</span>comparisons<span class="sc">$</span>diffExpressed <span class="sc">==</span> <span class="st">"Not sig"</span>,], <span class="fu">aes</span>(<span class="at">x=</span>comparison, <span class="at">fill=</span>diffExpressed)) <span class="sc">+</span> </span>
<span id="cb5-14"><a href="#cb5-14" aria-hidden="true" tabindex="-1"></a> <span class="fu">geom_bar</span>(<span class="at">stat=</span><span class="st">"count"</span>) <span class="sc">+</span> </span>
<span id="cb5-15"><a href="#cb5-15" aria-hidden="true" tabindex="-1"></a> <span class="fu">xlab</span>(<span class="st">""</span>) <span class="sc">+</span> </span>
<span id="cb5-16"><a href="#cb5-16" aria-hidden="true" tabindex="-1"></a> <span class="fu">ylab</span>(<span class="st">"Number of DEGs"</span>) <span class="sc">+</span></span>
<span id="cb5-17"><a href="#cb5-17" aria-hidden="true" tabindex="-1"></a> <span class="fu">scale_fill_manual</span>(<span class="at">values=</span><span class="fu">ghibli_palette</span>(<span class="st">"YesterdayLight"</span>, <span class="at">direction=</span><span class="sc">-</span><span class="dv">1</span>), <span class="at">labels=</span><span class="fu">c</span>(<span class="st">'Up'</span>, <span class="st">'Down'</span>), <span class="at">name=</span><span class="st">""</span>) <span class="sc">+</span> </span>
<span id="cb5-18"><a href="#cb5-18" aria-hidden="true" tabindex="-1"></a> <span class="fu">theme_bw</span>() <span class="sc">+</span> </span>
<span id="cb5-19"><a href="#cb5-19" aria-hidden="true" tabindex="-1"></a> <span class="fu">geom_text</span>(<span class="at">stat =</span> <span class="st">'count'</span>, <span class="fu">aes</span>(<span class="at">label=</span><span class="fu">after_stat</span>(count), <span class="at">y=</span><span class="fu">after_stat</span>(count) <span class="sc">+</span> <span class="dv">1</span>), <span class="at">position=</span><span class="fu">position_stack</span>(<span class="at">vjust=</span><span class="fl">0.5</span>), <span class="at">size=</span><span class="fl">6.5</span>, <span class="at">colour=</span><span class="st">"black"</span>) <span class="sc">+</span> </span>
<span id="cb5-20"><a href="#cb5-20" aria-hidden="true" tabindex="-1"></a> <span class="fu">theme</span>(<span class="at">legend.text=</span><span class="fu">element_text</span>(<span class="at">size=</span><span class="dv">20</span>), <span class="at">text =</span> <span class="fu">element_text</span>(<span class="at">size =</span> <span class="dv">20</span>))</span>
<span id="cb5-21"><a href="#cb5-21" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb5-22"><a href="#cb5-22" aria-hidden="true" tabindex="-1"></a><span class="fu">plot</span>(degs_plot)</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
</details>
<div class="cell-output-display">
<div class="quarto-figure quarto-figure-center">
<figure class="figure">
<p><img src="manuscript_figures_files/figure-html/figure_1d-1.png" class="img-fluid quarto-figure quarto-figure-center figure-img" width="672"></p>
</figure>
</div>
</div>
</div>
<p><strong>Figure 1d) Stacked barplot of the number of differentially expressed genes (DEGs; FDR < 0.05) identified through pairwise comparisons across the experimental groups.</strong></p>
<div class="cell" data-layout-align="center">
<details class="code-fold">
<summary>Code</summary>
<div class="sourceCode cell-code" id="cb6"><pre class="sourceCode r code-with-copy"><code class="sourceCode r"><span id="cb6-1"><a href="#cb6-1" aria-hidden="true" tabindex="-1"></a><span class="do">## Figure 1e: no of DARs with DEG overlap ## </span></span>
<span id="cb6-2"><a href="#cb6-2" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb6-3"><a href="#cb6-3" aria-hidden="true" tabindex="-1"></a><span class="co"># Function to get data gene names annotated to significant peaks</span></span>
<span id="cb6-4"><a href="#cb6-4" aria-hidden="true" tabindex="-1"></a>create_gene_df <span class="ot"><-</span> <span class="cf">function</span>(sig_data, symbol_col, diff_col, contrast_col, assay_name) {</span>
<span id="cb6-5"><a href="#cb6-5" aria-hidden="true" tabindex="-1"></a> gene_df <span class="ot"><-</span> sig_data[, <span class="fu">c</span>(symbol_col, diff_col, contrast_col)]</span>
<span id="cb6-6"><a href="#cb6-6" aria-hidden="true" tabindex="-1"></a> gene_df<span class="sc">$</span>assay <span class="ot"><-</span> assay_name</span>
<span id="cb6-7"><a href="#cb6-7" aria-hidden="true" tabindex="-1"></a> <span class="fu">colnames</span>(gene_df) <span class="ot"><-</span> <span class="fu">c</span>(<span class="st">"gene"</span>, <span class="st">"direction"</span>, <span class="st">"contrast"</span>, <span class="st">"assay"</span>)</span>
<span id="cb6-8"><a href="#cb6-8" aria-hidden="true" tabindex="-1"></a> <span class="fu">return</span>(gene_df)</span>
<span id="cb6-9"><a href="#cb6-9" aria-hidden="true" tabindex="-1"></a>}</span>
<span id="cb6-10"><a href="#cb6-10" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb6-11"><a href="#cb6-11" aria-hidden="true" tabindex="-1"></a><span class="co"># Create ATAC-seq gene data frames</span></span>
<span id="cb6-12"><a href="#cb6-12" aria-hidden="true" tabindex="-1"></a>E2vsE3_atac_genes <span class="ot"><-</span> <span class="fu">create_gene_df</span>(E2vsE3_atac_sig, <span class="st">"SYMBOL"</span>, <span class="st">"diffAccessible"</span>, <span class="st">"contrast"</span>, <span class="st">"ATAC-seq"</span>)</span>
<span id="cb6-13"><a href="#cb6-13" aria-hidden="true" tabindex="-1"></a>E4vsE3_atac_genes <span class="ot"><-</span> <span class="fu">create_gene_df</span>(E4vsE3_atac_sig, <span class="st">"SYMBOL"</span>, <span class="st">"diffAccessible"</span>, <span class="st">"contrast"</span>, <span class="st">"ATAC-seq"</span>)</span>
<span id="cb6-14"><a href="#cb6-14" aria-hidden="true" tabindex="-1"></a>E4vsE2_atac_genes <span class="ot"><-</span> <span class="fu">create_gene_df</span>(E4vsE2_atac_sig, <span class="st">"SYMBOL"</span>, <span class="st">"diffAccessible"</span>, <span class="st">"contrast"</span>, <span class="st">"ATAC-seq"</span>)</span>
<span id="cb6-15"><a href="#cb6-15" aria-hidden="true" tabindex="-1"></a>KOvsE3_atac_genes <span class="ot"><-</span> <span class="fu">create_gene_df</span>(KOvsE3_atac_sig, <span class="st">"SYMBOL"</span>, <span class="st">"diffAccessible"</span>, <span class="st">"contrast"</span>, <span class="st">"ATAC-seq"</span>)</span>
<span id="cb6-16"><a href="#cb6-16" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb6-17"><a href="#cb6-17" aria-hidden="true" tabindex="-1"></a><span class="co"># Combine ATAC-seq gene data frames</span></span>
<span id="cb6-18"><a href="#cb6-18" aria-hidden="true" tabindex="-1"></a>atac_genes <span class="ot"><-</span> <span class="fu">rbind</span>(E2vsE3_atac_genes, E4vsE3_atac_genes, E4vsE2_atac_genes, KOvsE3_atac_genes)</span>
<span id="cb6-19"><a href="#cb6-19" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb6-20"><a href="#cb6-20" aria-hidden="true" tabindex="-1"></a><span class="co"># Create RNA-seq gene data frames</span></span>
<span id="cb6-21"><a href="#cb6-21" aria-hidden="true" tabindex="-1"></a>E2vsE3_rna_genes <span class="ot"><-</span> <span class="fu">create_gene_df</span>(comparisons[comparisons<span class="sc">$</span>fdr <span class="sc"><</span> <span class="fl">0.05</span> <span class="sc">&</span> comparisons<span class="sc">$</span>comparison <span class="sc">==</span> <span class="st">"E2vsE3"</span>,], <span class="st">"hgnc_symbol"</span>, <span class="st">"diffExpressed"</span>, <span class="st">"comparison"</span>, <span class="st">"RNA-seq"</span>)</span>
<span id="cb6-22"><a href="#cb6-22" aria-hidden="true" tabindex="-1"></a>E4vsE3_rna_genes <span class="ot"><-</span> <span class="fu">create_gene_df</span>(comparisons[comparisons<span class="sc">$</span>fdr <span class="sc"><</span> <span class="fl">0.05</span> <span class="sc">&</span> comparisons<span class="sc">$</span>comparison <span class="sc">==</span> <span class="st">"E4vsE3"</span>,], <span class="st">"hgnc_symbol"</span>, <span class="st">"diffExpressed"</span>, <span class="st">"comparison"</span>, <span class="st">"RNA-seq"</span>)</span>
<span id="cb6-23"><a href="#cb6-23" aria-hidden="true" tabindex="-1"></a>E4vsE2_rna_genes <span class="ot"><-</span> <span class="fu">create_gene_df</span>(comparisons[comparisons<span class="sc">$</span>fdr <span class="sc"><</span> <span class="fl">0.05</span> <span class="sc">&</span> comparisons<span class="sc">$</span>comparison <span class="sc">==</span> <span class="st">"E4vsE2"</span>,], <span class="st">"hgnc_symbol"</span>, <span class="st">"diffExpressed"</span>, <span class="st">"comparison"</span>, <span class="st">"RNA-seq"</span>)</span>
<span id="cb6-24"><a href="#cb6-24" aria-hidden="true" tabindex="-1"></a>KOvsE3_rna_genes <span class="ot"><-</span> <span class="fu">create_gene_df</span>(comparisons[comparisons<span class="sc">$</span>fdr <span class="sc"><</span> <span class="fl">0.05</span> <span class="sc">&</span> comparisons<span class="sc">$</span>comparison <span class="sc">==</span> <span class="st">"KOvsE3"</span>,], <span class="st">"hgnc_symbol"</span>, <span class="st">"diffExpressed"</span>, <span class="st">"comparison"</span>, <span class="st">"RNA-seq"</span>)</span>
<span id="cb6-25"><a href="#cb6-25" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb6-26"><a href="#cb6-26" aria-hidden="true" tabindex="-1"></a><span class="co"># Combine RNA-seq gene data frames</span></span>
<span id="cb6-27"><a href="#cb6-27" aria-hidden="true" tabindex="-1"></a>rna_genes <span class="ot"><-</span> <span class="fu">rbind</span>(E2vsE3_rna_genes, E4vsE3_rna_genes, E4vsE2_rna_genes, KOvsE3_rna_genes)</span>
<span id="cb6-28"><a href="#cb6-28" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb6-29"><a href="#cb6-29" aria-hidden="true" tabindex="-1"></a><span class="co"># Combine RNA-seq and ATAC-seq gene data frames</span></span>
<span id="cb6-30"><a href="#cb6-30" aria-hidden="true" tabindex="-1"></a>rna_atac_genes <span class="ot"><-</span> <span class="fu">rbind</span>(atac_genes, rna_genes)</span>
<span id="cb6-31"><a href="#cb6-31" aria-hidden="true" tabindex="-1"></a>rna_atac_genes<span class="sc">$</span>direction <span class="ot"><-</span> <span class="fu">gsub</span>(<span class="st">"Upregulated"</span>, <span class="st">"Up"</span>, rna_atac_genes<span class="sc">$</span>direction)</span>
<span id="cb6-32"><a href="#cb6-32" aria-hidden="true" tabindex="-1"></a>rna_atac_genes<span class="sc">$</span>direction <span class="ot"><-</span> <span class="fu">gsub</span>(<span class="st">"Downregulated"</span>, <span class="st">"Down"</span>, rna_atac_genes<span class="sc">$</span>direction)</span>
<span id="cb6-33"><a href="#cb6-33" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb6-34"><a href="#cb6-34" aria-hidden="true" tabindex="-1"></a><span class="co"># Summarise gene counts</span></span>
<span id="cb6-35"><a href="#cb6-35" aria-hidden="true" tabindex="-1"></a>gene_counts <span class="ot"><-</span> rna_atac_genes <span class="sc">%>%</span></span>
<span id="cb6-36"><a href="#cb6-36" aria-hidden="true" tabindex="-1"></a> <span class="fu">filter</span>(gene <span class="sc">!=</span> <span class="st">""</span>) <span class="sc">%>%</span></span>
<span id="cb6-37"><a href="#cb6-37" aria-hidden="true" tabindex="-1"></a> <span class="fu">group_by</span>(contrast, assay) <span class="sc">%>%</span></span>
<span id="cb6-38"><a href="#cb6-38" aria-hidden="true" tabindex="-1"></a> dplyr<span class="sc">::</span><span class="fu">summarise</span>(<span class="at">count =</span> <span class="fu">n</span>(), <span class="at">.groups =</span> <span class="st">'drop'</span>)</span>
<span id="cb6-39"><a href="#cb6-39" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb6-40"><a href="#cb6-40" aria-hidden="true" tabindex="-1"></a><span class="co"># Count the number of overlapping genes</span></span>
<span id="cb6-41"><a href="#cb6-41" aria-hidden="true" tabindex="-1"></a>overlap_counts <span class="ot"><-</span> rna_atac_genes <span class="sc">%>%</span></span>
<span id="cb6-42"><a href="#cb6-42" aria-hidden="true" tabindex="-1"></a> <span class="fu">filter</span>(gene <span class="sc">!=</span> <span class="st">""</span>) <span class="sc">%>%</span></span>
<span id="cb6-43"><a href="#cb6-43" aria-hidden="true" tabindex="-1"></a> <span class="fu">group_by</span>(contrast, gene) <span class="sc">%>%</span></span>
<span id="cb6-44"><a href="#cb6-44" aria-hidden="true" tabindex="-1"></a> dplyr<span class="sc">::</span><span class="fu">summarise</span>(<span class="at">count =</span> <span class="fu">n</span>(), <span class="at">.groups =</span> <span class="st">'drop'</span>) <span class="sc">%>%</span></span>
<span id="cb6-45"><a href="#cb6-45" aria-hidden="true" tabindex="-1"></a> <span class="fu">filter</span>(count <span class="sc">></span> <span class="dv">1</span>) <span class="sc">%>%</span></span>
<span id="cb6-46"><a href="#cb6-46" aria-hidden="true" tabindex="-1"></a> <span class="fu">group_by</span>(contrast) <span class="sc">%>%</span></span>
<span id="cb6-47"><a href="#cb6-47" aria-hidden="true" tabindex="-1"></a> dplyr<span class="sc">::</span><span class="fu">summarise</span>(<span class="at">overlap_count =</span> <span class="fu">n</span>(), <span class="at">.groups =</span> <span class="st">'drop'</span>)</span>
<span id="cb6-48"><a href="#cb6-48" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb6-49"><a href="#cb6-49" aria-hidden="true" tabindex="-1"></a><span class="co"># Merge gene counts with overlap counts</span></span>
<span id="cb6-50"><a href="#cb6-50" aria-hidden="true" tabindex="-1"></a>gene_counts <span class="ot"><-</span> dplyr<span class="sc">::</span><span class="fu">left_join</span>(gene_counts, overlap_counts, <span class="at">by =</span> <span class="st">"contrast"</span>)</span>
<span id="cb6-51"><a href="#cb6-51" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb6-52"><a href="#cb6-52" aria-hidden="true" tabindex="-1"></a><span class="co"># Plot the results</span></span>
<span id="cb6-53"><a href="#cb6-53" aria-hidden="true" tabindex="-1"></a>dars_plot <span class="ot"><-</span> <span class="fu">ggplot</span>(gene_counts[gene_counts<span class="sc">$</span>assay <span class="sc">==</span> <span class="st">"ATAC-seq"</span>, ], <span class="fu">aes</span>(<span class="at">x =</span> contrast, <span class="at">y =</span> count, <span class="at">fill =</span> assay)) <span class="sc">+</span></span>
<span id="cb6-54"><a href="#cb6-54" aria-hidden="true" tabindex="-1"></a> <span class="fu">geom_bar</span>(<span class="at">stat =</span> <span class="st">"identity"</span>, <span class="at">position =</span> <span class="st">"dodge"</span>) <span class="sc">+</span></span>
<span id="cb6-55"><a href="#cb6-55" aria-hidden="true" tabindex="-1"></a> <span class="fu">geom_bar</span>(<span class="fu">aes</span>(<span class="at">y =</span> overlap_count, <span class="at">fill =</span> <span class="st">"Overlap"</span>), <span class="at">stat =</span> <span class="st">"identity"</span>, <span class="at">position =</span> <span class="st">"dodge"</span>) <span class="sc">+</span></span>
<span id="cb6-56"><a href="#cb6-56" aria-hidden="true" tabindex="-1"></a> <span class="fu">geom_text</span>(<span class="fu">aes</span>(<span class="at">label =</span> <span class="fu">paste</span>(overlap_count, <span class="st">"/"</span>, count)), <span class="at">position =</span> <span class="fu">position_dodge</span>(<span class="at">width =</span> <span class="fl">0.9</span>), <span class="at">vjust =</span> <span class="sc">-</span><span class="fl">0.5</span>, <span class="at">size =</span> <span class="fl">4.5</span>) <span class="sc">+</span></span>
<span id="cb6-57"><a href="#cb6-57" aria-hidden="true" tabindex="-1"></a> <span class="fu">labs</span>(<span class="at">x =</span> <span class="st">""</span>, <span class="at">y =</span> <span class="st">"Number of DARs"</span>, <span class="at">fill =</span> <span class="st">"Assay"</span>) <span class="sc">+</span></span>
<span id="cb6-58"><a href="#cb6-58" aria-hidden="true" tabindex="-1"></a> <span class="fu">ylim</span>(<span class="dv">0</span>, <span class="dv">80</span>) <span class="sc">+</span></span>
<span id="cb6-59"><a href="#cb6-59" aria-hidden="true" tabindex="-1"></a> <span class="fu">scale_fill_manual</span>(<span class="at">values =</span> <span class="fu">ghibli_palette</span>(<span class="st">"MarnieLight2"</span>, <span class="at">direction =</span> <span class="sc">-</span><span class="dv">1</span>), <span class="at">labels =</span> <span class="fu">c</span>(<span class="st">'ATAC only'</span>, <span class="st">'ATAC and RNA'</span>), <span class="at">name =</span> <span class="st">""</span>) <span class="sc">+</span></span>
<span id="cb6-60"><a href="#cb6-60" aria-hidden="true" tabindex="-1"></a> <span class="fu">theme_bw</span>() <span class="sc">+</span></span>
<span id="cb6-61"><a href="#cb6-61" aria-hidden="true" tabindex="-1"></a> <span class="fu">theme</span>(<span class="at">legend.text =</span> <span class="fu">element_text</span>(<span class="at">size =</span> <span class="dv">20</span>), <span class="at">text =</span> <span class="fu">element_text</span>(<span class="at">size =</span> <span class="dv">20</span>))</span>
<span id="cb6-62"><a href="#cb6-62" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb6-63"><a href="#cb6-63" aria-hidden="true" tabindex="-1"></a><span class="fu">plot</span>(dars_plot)</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
</details>
<div class="cell-output-display">
<div class="quarto-figure quarto-figure-center">
<figure class="figure">
<p><img src="manuscript_figures_files/figure-html/figure_1e-1.png" class="img-fluid quarto-figure quarto-figure-center figure-img" width="672"></p>
</figure>
</div>
</div>
</div>
<p><strong>Figure 1e) Stacked barplot of the number of differentially accessible regions (DARs) and how many of these overlap with the DEGs based on peak-to-gene annotation.</strong></p>
<p><strong>Figure 2: Microglia-expressing the different APOE isoforms exhibit widespread differences in gene regulation.</strong></p>
<div class="cell" data-layout-align="center">
<details class="code-fold">
<summary>Code</summary>
<div class="sourceCode cell-code" id="cb7"><pre class="sourceCode r code-with-copy"><code class="sourceCode r"><span id="cb7-1"><a href="#cb7-1" aria-hidden="true" tabindex="-1"></a><span class="do">## Figure 2s: E2vsE3 DEGs volcano plot ##</span></span>
<span id="cb7-2"><a href="#cb7-2" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb7-3"><a href="#cb7-3" aria-hidden="true" tabindex="-1"></a><span class="co"># set factor level </span></span>
<span id="cb7-4"><a href="#cb7-4" aria-hidden="true" tabindex="-1"></a>comparisons<span class="sc">$</span>diffExpressed <span class="ot"><-</span> <span class="fu">factor</span>(comparisons<span class="sc">$</span>diffExpressed, <span class="at">levels=</span><span class="fu">c</span>(<span class="st">"Up"</span>, <span class="st">"Down"</span>, <span class="st">"Not sig"</span>))</span>
<span id="cb7-5"><a href="#cb7-5" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb7-6"><a href="#cb7-6" aria-hidden="true" tabindex="-1"></a><span class="co"># fill in missing gene symbol for one of the top sig genes</span></span>
<span id="cb7-7"><a href="#cb7-7" aria-hidden="true" tabindex="-1"></a>comparisons<span class="sc">$</span>hgnc_symbol[comparisons<span class="sc">$</span>entrezgene_id<span class="sc">==</span><span class="st">"102724316"</span>] <span class="ot"><-</span> <span class="st">"SVIL-AS1"</span></span>
<span id="cb7-8"><a href="#cb7-8" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb7-9"><a href="#cb7-9" aria-hidden="true" tabindex="-1"></a><span class="co"># get top 20 genes for labelling </span></span>
<span id="cb7-10"><a href="#cb7-10" aria-hidden="true" tabindex="-1"></a>top_genes <span class="ot"><-</span> <span class="fu">head</span>(comparisons[comparisons<span class="sc">$</span>comparison <span class="sc">==</span> <span class="st">"E2vsE3"</span>, ], <span class="dv">10</span>)</span>
<span id="cb7-11"><a href="#cb7-11" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb7-12"><a href="#cb7-12" aria-hidden="true" tabindex="-1"></a>E2vsE3_volcano <span class="ot"><-</span> <span class="fu">ggplot</span>(comparisons[comparisons<span class="sc">$</span>comparison<span class="sc">==</span><span class="st">"E2vsE3"</span>,], </span>
<span id="cb7-13"><a href="#cb7-13" aria-hidden="true" tabindex="-1"></a> <span class="fu">aes</span>(<span class="at">x=</span>log2FoldChange, <span class="at">y=</span><span class="sc">-</span><span class="fu">log10</span>(fdr), <span class="at">colour=</span>diffExpressed)) <span class="sc">+</span> </span>
<span id="cb7-14"><a href="#cb7-14" aria-hidden="true" tabindex="-1"></a> <span class="fu">geom_point</span>(<span class="at">size=</span><span class="dv">1</span>) <span class="sc">+</span> </span>
<span id="cb7-15"><a href="#cb7-15" aria-hidden="true" tabindex="-1"></a> <span class="fu">xlim</span>(<span class="sc">-</span><span class="dv">10</span>,<span class="dv">10</span>) <span class="sc">+</span> </span>
<span id="cb7-16"><a href="#cb7-16" aria-hidden="true" tabindex="-1"></a> <span class="fu">scale_colour_manual</span>(<span class="at">values=</span><span class="fu">c</span>(<span class="st">"#92BBD9FF"</span>, <span class="st">"#92BBD9FF"</span>, <span class="st">"#C3DAEAFF"</span>)) <span class="sc">+</span> </span>
<span id="cb7-17"><a href="#cb7-17" aria-hidden="true" tabindex="-1"></a> <span class="fu">theme_classic</span>() <span class="sc">+</span> </span>
<span id="cb7-18"><a href="#cb7-18" aria-hidden="true" tabindex="-1"></a> <span class="fu">ylab</span>(<span class="st">"-log10(FDR)"</span>) <span class="sc">+</span> </span>
<span id="cb7-19"><a href="#cb7-19" aria-hidden="true" tabindex="-1"></a> <span class="fu">guides</span>(<span class="at">colour=</span><span class="st">"none"</span>) <span class="sc">+</span> </span>
<span id="cb7-20"><a href="#cb7-20" aria-hidden="true" tabindex="-1"></a> <span class="fu">theme</span>(<span class="at">text =</span> <span class="fu">element_text</span>(<span class="at">size=</span><span class="dv">16</span>), <span class="at">plot.title =</span> <span class="fu">element_text</span>(<span class="at">hjust =</span> <span class="fl">0.5</span>)) <span class="sc">+</span> </span>
<span id="cb7-21"><a href="#cb7-21" aria-hidden="true" tabindex="-1"></a> <span class="fu">ggtitle</span>(<span class="st">"E2 vs E3"</span>) <span class="sc">+</span> </span>
<span id="cb7-22"><a href="#cb7-22" aria-hidden="true" tabindex="-1"></a> <span class="fu">geom_label_repel</span>(<span class="at">data =</span> top_genes, <span class="fu">aes</span>(<span class="at">label =</span> hgnc_symbol), <span class="at">box.padding =</span> <span class="fl">0.5</span>, <span class="at">colour=</span><span class="st">"#506777FF"</span>, <span class="at">size=</span><span class="dv">5</span>) <span class="sc">+</span></span>
<span id="cb7-23"><a href="#cb7-23" aria-hidden="true" tabindex="-1"></a> <span class="fu">geom_vline</span>(<span class="at">xintercept =</span> <span class="dv">0</span>, <span class="at">linetype =</span> <span class="st">"dashed"</span>, <span class="at">color =</span> <span class="st">"slategrey"</span>) <span class="sc">+</span></span>
<span id="cb7-24"><a href="#cb7-24" aria-hidden="true" tabindex="-1"></a> <span class="fu">geom_hline</span>(<span class="at">yintercept =</span> <span class="sc">-</span><span class="fu">log10</span>(<span class="fl">0.05</span>), <span class="at">linetype =</span> <span class="st">"dashed"</span>, <span class="at">color =</span> <span class="st">"slategrey"</span>)</span>
<span id="cb7-25"><a href="#cb7-25" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb7-26"><a href="#cb7-26" aria-hidden="true" tabindex="-1"></a><span class="fu">plot</span>(E2vsE3_volcano)</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
</details>
<div class="cell-output-display">
<div class="quarto-figure quarto-figure-center">
<figure class="figure">
<p><img src="manuscript_figures_files/figure-html/figure_2a-1.png" class="img-fluid quarto-figure quarto-figure-center figure-img" width="576"></p>
</figure>
</div>
</div>
</div>
<p><strong>Figure 2a) Differentially expressed genes in <em>APOE2</em> vs <em>APOE3</em>-expressing microglia.</strong></p>
<div class="cell" data-layout-align="center">
<details class="code-fold">
<summary>Code</summary>
<div class="sourceCode cell-code" id="cb8"><pre class="sourceCode r code-with-copy"><code class="sourceCode r"><span id="cb8-1"><a href="#cb8-1" aria-hidden="true" tabindex="-1"></a><span class="do">## Figure 2b: E4vsE3 DEGs volcano plot ##</span></span>
<span id="cb8-2"><a href="#cb8-2" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb8-3"><a href="#cb8-3" aria-hidden="true" tabindex="-1"></a><span class="co"># get top 20 genes for labelling </span></span>
<span id="cb8-4"><a href="#cb8-4" aria-hidden="true" tabindex="-1"></a>top_genes <span class="ot"><-</span> <span class="fu">head</span>(comparisons[comparisons<span class="sc">$</span>comparison <span class="sc">==</span> <span class="st">"E4vsE3"</span>, ], <span class="dv">10</span>)</span>
<span id="cb8-5"><a href="#cb8-5" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb8-6"><a href="#cb8-6" aria-hidden="true" tabindex="-1"></a>E4vsE3_volcano <span class="ot"><-</span> <span class="fu">ggplot</span>(comparisons[comparisons<span class="sc">$</span>comparison<span class="sc">==</span><span class="st">"E4vsE3"</span>,], </span>
<span id="cb8-7"><a href="#cb8-7" aria-hidden="true" tabindex="-1"></a> <span class="fu">aes</span>(<span class="at">x=</span>log2FoldChange, <span class="at">y=</span><span class="sc">-</span><span class="fu">log10</span>(fdr), <span class="at">colour=</span>diffExpressed)) <span class="sc">+</span> </span>
<span id="cb8-8"><a href="#cb8-8" aria-hidden="true" tabindex="-1"></a> <span class="fu">geom_point</span>(<span class="at">size=</span><span class="dv">1</span>) <span class="sc">+</span> </span>
<span id="cb8-9"><a href="#cb8-9" aria-hidden="true" tabindex="-1"></a> <span class="fu">xlim</span>(<span class="sc">-</span><span class="dv">10</span>,<span class="dv">10</span>) <span class="sc">+</span> </span>
<span id="cb8-10"><a href="#cb8-10" aria-hidden="true" tabindex="-1"></a> <span class="fu">scale_colour_manual</span>(<span class="at">values=</span><span class="fu">c</span>(<span class="st">"#DCCA2CFF"</span>, <span class="st">"#DCCA2CFF"</span>, <span class="st">"#ECE28BFF"</span>)) <span class="sc">+</span> </span>
<span id="cb8-11"><a href="#cb8-11" aria-hidden="true" tabindex="-1"></a> <span class="fu">theme_classic</span>() <span class="sc">+</span> </span>
<span id="cb8-12"><a href="#cb8-12" aria-hidden="true" tabindex="-1"></a> <span class="fu">ylab</span>(<span class="st">"-log10(FDR)"</span>) <span class="sc">+</span> </span>
<span id="cb8-13"><a href="#cb8-13" aria-hidden="true" tabindex="-1"></a> <span class="fu">guides</span>(<span class="at">colour=</span><span class="st">"none"</span>) <span class="sc">+</span> </span>
<span id="cb8-14"><a href="#cb8-14" aria-hidden="true" tabindex="-1"></a> <span class="fu">theme</span>(<span class="at">text =</span> <span class="fu">element_text</span>(<span class="at">size=</span><span class="dv">16</span>), <span class="at">plot.title =</span> <span class="fu">element_text</span>(<span class="at">hjust =</span> <span class="fl">0.5</span>)) <span class="sc">+</span> </span>
<span id="cb8-15"><a href="#cb8-15" aria-hidden="true" tabindex="-1"></a> <span class="fu">ggtitle</span>(<span class="st">"E4 vs E3"</span>) <span class="sc">+</span> </span>
<span id="cb8-16"><a href="#cb8-16" aria-hidden="true" tabindex="-1"></a> <span class="fu">geom_label_repel</span>(<span class="at">data =</span> top_genes, <span class="fu">aes</span>(<span class="at">label =</span> hgnc_symbol), <span class="at">box.padding =</span> <span class="fl">0.5</span>, <span class="at">colour=</span><span class="st">"#796F18FF"</span>, <span class="at">size=</span><span class="dv">5</span>) <span class="sc">+</span></span>
<span id="cb8-17"><a href="#cb8-17" aria-hidden="true" tabindex="-1"></a> <span class="fu">geom_vline</span>(<span class="at">xintercept =</span> <span class="dv">0</span>, <span class="at">linetype =</span> <span class="st">"dashed"</span>, <span class="at">color =</span> <span class="st">"slategrey"</span>) <span class="sc">+</span></span>
<span id="cb8-18"><a href="#cb8-18" aria-hidden="true" tabindex="-1"></a> <span class="fu">geom_hline</span>(<span class="at">yintercept =</span> <span class="sc">-</span><span class="fu">log10</span>(<span class="fl">0.05</span>), <span class="at">linetype =</span> <span class="st">"dashed"</span>, <span class="at">color =</span> <span class="st">"slategrey"</span>)</span>
<span id="cb8-19"><a href="#cb8-19" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb8-20"><a href="#cb8-20" aria-hidden="true" tabindex="-1"></a><span class="fu">plot</span>(E4vsE3_volcano)</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
</details>
<div class="cell-output-display">
<div class="quarto-figure quarto-figure-center">
<figure class="figure">
<p><img src="manuscript_figures_files/figure-html/figure_2b-1.png" class="img-fluid quarto-figure quarto-figure-center figure-img" width="576"></p>
</figure>
</div>
</div>
</div>
<p><strong>Figure 2b) Differentially expressed genes in <em>APOE4</em> vs <em>APOE3</em>-expressing microglia.</strong></p>
<div class="cell" data-layout-align="center">
<details class="code-fold">
<summary>Code</summary>
<div class="sourceCode cell-code" id="cb9"><pre class="sourceCode r code-with-copy"><code class="sourceCode r"><span id="cb9-1"><a href="#cb9-1" aria-hidden="true" tabindex="-1"></a><span class="do">## Figure 2c: E4vsE2 DEGs volcano plot ##</span></span>
<span id="cb9-2"><a href="#cb9-2" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb9-3"><a href="#cb9-3" aria-hidden="true" tabindex="-1"></a><span class="co"># get top 20 genes for labelling </span></span>
<span id="cb9-4"><a href="#cb9-4" aria-hidden="true" tabindex="-1"></a>top_genes <span class="ot"><-</span> <span class="fu">head</span>(comparisons[comparisons<span class="sc">$</span>comparison <span class="sc">==</span> <span class="st">"E4vsE2"</span>, ], <span class="dv">10</span>)</span>
<span id="cb9-5"><a href="#cb9-5" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb9-6"><a href="#cb9-6" aria-hidden="true" tabindex="-1"></a>E4vsE2_volcano <span class="ot"><-</span> <span class="fu">ggplot</span>(comparisons[comparisons<span class="sc">$</span>comparison<span class="sc">==</span><span class="st">"E4vsE2"</span>,], </span>
<span id="cb9-7"><a href="#cb9-7" aria-hidden="true" tabindex="-1"></a> <span class="fu">aes</span>(<span class="at">x=</span>log2FoldChange, <span class="at">y=</span><span class="sc">-</span><span class="fu">log10</span>(fdr), <span class="at">colour=</span>diffExpressed)) <span class="sc">+</span> </span>
<span id="cb9-8"><a href="#cb9-8" aria-hidden="true" tabindex="-1"></a> <span class="fu">geom_point</span>(<span class="at">size=</span><span class="dv">1</span>) <span class="sc">+</span> </span>
<span id="cb9-9"><a href="#cb9-9" aria-hidden="true" tabindex="-1"></a> <span class="fu">xlim</span>(<span class="sc">-</span><span class="dv">10</span>,<span class="dv">10</span>) <span class="sc">+</span> </span>
<span id="cb9-10"><a href="#cb9-10" aria-hidden="true" tabindex="-1"></a> <span class="fu">scale_colour_manual</span>(<span class="at">values=</span><span class="fu">c</span>(<span class="st">"#6FB382FF"</span>, <span class="st">"#6FB382FF"</span>, <span class="st">"#B1D5BBFF"</span>)) <span class="sc">+</span> </span>
<span id="cb9-11"><a href="#cb9-11" aria-hidden="true" tabindex="-1"></a> <span class="fu">theme_classic</span>() <span class="sc">+</span> </span>
<span id="cb9-12"><a href="#cb9-12" aria-hidden="true" tabindex="-1"></a> <span class="fu">ylab</span>(<span class="st">"-log10(FDR)"</span>) <span class="sc">+</span> </span>
<span id="cb9-13"><a href="#cb9-13" aria-hidden="true" tabindex="-1"></a> <span class="fu">guides</span>(<span class="at">colour=</span><span class="st">"none"</span>) <span class="sc">+</span> </span>
<span id="cb9-14"><a href="#cb9-14" aria-hidden="true" tabindex="-1"></a> <span class="fu">theme</span>(<span class="at">text =</span> <span class="fu">element_text</span>(<span class="at">size=</span><span class="dv">16</span>), <span class="at">plot.title =</span> <span class="fu">element_text</span>(<span class="at">hjust =</span> <span class="fl">0.5</span>)) <span class="sc">+</span> </span>
<span id="cb9-15"><a href="#cb9-15" aria-hidden="true" tabindex="-1"></a> <span class="fu">ggtitle</span>(<span class="st">"E4 vs E2"</span>) <span class="sc">+</span> </span>
<span id="cb9-16"><a href="#cb9-16" aria-hidden="true" tabindex="-1"></a> <span class="fu">geom_label_repel</span>(<span class="at">data =</span> top_genes, <span class="fu">aes</span>(<span class="at">label =</span> hgnc_symbol), <span class="at">box.padding =</span> <span class="fl">0.5</span>, <span class="at">colour=</span><span class="st">"#3E6248FF"</span>, <span class="at">size=</span><span class="dv">5</span>) <span class="sc">+</span></span>
<span id="cb9-17"><a href="#cb9-17" aria-hidden="true" tabindex="-1"></a> <span class="fu">geom_vline</span>(<span class="at">xintercept =</span> <span class="dv">0</span>, <span class="at">linetype =</span> <span class="st">"dashed"</span>, <span class="at">color =</span> <span class="st">"slategrey"</span>) <span class="sc">+</span></span>
<span id="cb9-18"><a href="#cb9-18" aria-hidden="true" tabindex="-1"></a> <span class="fu">geom_hline</span>(<span class="at">yintercept =</span> <span class="sc">-</span><span class="fu">log10</span>(<span class="fl">0.05</span>), <span class="at">linetype =</span> <span class="st">"dashed"</span>, <span class="at">color =</span> <span class="st">"slategrey"</span>)</span>
<span id="cb9-19"><a href="#cb9-19" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb9-20"><a href="#cb9-20" aria-hidden="true" tabindex="-1"></a><span class="fu">plot</span>(E4vsE2_volcano)</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
</details>
<div class="cell-output-display">
<div class="quarto-figure quarto-figure-center">
<figure class="figure">
<p><img src="manuscript_figures_files/figure-html/figure_2c-1.png" class="img-fluid quarto-figure quarto-figure-center figure-img" width="576"></p>
</figure>
</div>
</div>
</div>
<p><strong>Figure 2c) Differentially expressed genes in <em>APOE4</em> vs <em>APOE2</em>-expressing microglia.</strong></p>
<div class="cell" data-layout-align="center">
<details class="code-fold">
<summary>Code</summary>
<div class="sourceCode cell-code" id="cb10"><pre class="sourceCode r code-with-copy"><code class="sourceCode r"><span id="cb10-1"><a href="#cb10-1" aria-hidden="true" tabindex="-1"></a><span class="do">## Figure 2d: RRHO E2 and KO ##</span></span>
<span id="cb10-2"><a href="#cb10-2" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb10-3"><a href="#cb10-3" aria-hidden="true" tabindex="-1"></a><span class="fu">set.seed</span>(<span class="dv">1234</span>)</span>
<span id="cb10-4"><a href="#cb10-4" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb10-5"><a href="#cb10-5" aria-hidden="true" tabindex="-1"></a><span class="co"># Set rrho p-value for KO vs E3</span></span>
<span id="cb10-6"><a href="#cb10-6" aria-hidden="true" tabindex="-1"></a>KOvsE3_rrho <span class="ot"><-</span> KOvsE3_rna[, <span class="fu">c</span>(<span class="st">"entrezgene_id"</span>, <span class="st">"pvalue"</span>, <span class="st">"log2FoldChange"</span>)]</span>
<span id="cb10-7"><a href="#cb10-7" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb10-8"><a href="#cb10-8" aria-hidden="true" tabindex="-1"></a>KOvsE3_rrho<span class="sc">$</span>rrho_pvalue <span class="ot"><-</span> <span class="dv">0</span></span>
<span id="cb10-9"><a href="#cb10-9" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb10-10"><a href="#cb10-10" aria-hidden="true" tabindex="-1"></a>up_indices <span class="ot"><-</span> KOvsE3_rrho<span class="sc">$</span>log2FoldChange <span class="sc">></span> <span class="dv">0</span></span>
<span id="cb10-11"><a href="#cb10-11" aria-hidden="true" tabindex="-1"></a>down_indices <span class="ot"><-</span> KOvsE3_rrho<span class="sc">$</span>log2FoldChange <span class="sc"><</span> <span class="dv">0</span></span>
<span id="cb10-12"><a href="#cb10-12" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb10-13"><a href="#cb10-13" aria-hidden="true" tabindex="-1"></a>KOvsE3_rrho<span class="sc">$</span>rrho_pvalue[up_indices] <span class="ot"><-</span> <span class="sc">-</span><span class="fu">log10</span>(KOvsE3_rrho<span class="sc">$</span>pvalue[up_indices]) <span class="sc">*</span> KOvsE3_rrho<span class="sc">$</span>log2FoldChange[up_indices]</span>
<span id="cb10-14"><a href="#cb10-14" aria-hidden="true" tabindex="-1"></a>KOvsE3_rrho<span class="sc">$</span>rrho_pvalue[down_indices] <span class="ot"><-</span> <span class="sc">-</span><span class="fu">log10</span>(KOvsE3_rrho<span class="sc">$</span>pvalue[down_indices]) <span class="sc">*</span> KOvsE3_rrho<span class="sc">$</span>log2FoldChange[down_indices]</span>
<span id="cb10-15"><a href="#cb10-15" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb10-16"><a href="#cb10-16" aria-hidden="true" tabindex="-1"></a>KOvsE3_rrho <span class="ot"><-</span> KOvsE3_rrho[,<span class="fu">c</span>(<span class="st">"entrezgene_id"</span>, <span class="st">"rrho_pvalue"</span>)]</span>
<span id="cb10-17"><a href="#cb10-17" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb10-18"><a href="#cb10-18" aria-hidden="true" tabindex="-1"></a><span class="co"># Set rrho p-value for E2 vs E3</span></span>
<span id="cb10-19"><a href="#cb10-19" aria-hidden="true" tabindex="-1"></a>E2vsE3_rrho <span class="ot"><-</span> E2vsE3_rna[, <span class="fu">c</span>(<span class="st">"entrezgene_id"</span>, <span class="st">"pvalue"</span>, <span class="st">"log2FoldChange"</span>)]</span>
<span id="cb10-20"><a href="#cb10-20" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb10-21"><a href="#cb10-21" aria-hidden="true" tabindex="-1"></a>E2vsE3_rrho<span class="sc">$</span>rrho_pvalue <span class="ot"><-</span> <span class="dv">0</span></span>
<span id="cb10-22"><a href="#cb10-22" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb10-23"><a href="#cb10-23" aria-hidden="true" tabindex="-1"></a>up_indices <span class="ot"><-</span> E2vsE3_rrho<span class="sc">$</span>log2FoldChange <span class="sc">></span> <span class="dv">0</span></span>
<span id="cb10-24"><a href="#cb10-24" aria-hidden="true" tabindex="-1"></a>down_indices <span class="ot"><-</span> E2vsE3_rrho<span class="sc">$</span>log2FoldChange <span class="sc"><</span> <span class="dv">0</span></span>
<span id="cb10-25"><a href="#cb10-25" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb10-26"><a href="#cb10-26" aria-hidden="true" tabindex="-1"></a>E2vsE3_rrho<span class="sc">$</span>rrho_pvalue[up_indices] <span class="ot"><-</span> <span class="sc">-</span><span class="fu">log10</span>(E2vsE3_rrho<span class="sc">$</span>pvalue[up_indices]) <span class="sc">*</span> E2vsE3_rrho<span class="sc">$</span>log2FoldChange[up_indices]</span>
<span id="cb10-27"><a href="#cb10-27" aria-hidden="true" tabindex="-1"></a>E2vsE3_rrho<span class="sc">$</span>rrho_pvalue[down_indices] <span class="ot"><-</span> <span class="sc">-</span><span class="fu">log10</span>(E2vsE3_rrho<span class="sc">$</span>pvalue[down_indices]) <span class="sc">*</span> E2vsE3_rrho<span class="sc">$</span>log2FoldChange[down_indices]</span>
<span id="cb10-28"><a href="#cb10-28" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb10-29"><a href="#cb10-29" aria-hidden="true" tabindex="-1"></a>E2vsE3_rrho <span class="ot"><-</span> E2vsE3_rrho[,<span class="fu">c</span>(<span class="st">"entrezgene_id"</span>, <span class="st">"rrho_pvalue"</span>)]</span>
<span id="cb10-30"><a href="#cb10-30" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb10-31"><a href="#cb10-31" aria-hidden="true" tabindex="-1"></a>E2_KO_rrho <span class="ot"><-</span> <span class="fu">RRHO2_initialize</span>(<span class="fu">na.omit</span>(E2vsE3_rrho), <span class="fu">na.omit</span>(KOvsE3_rrho), <span class="at">labels =</span> <span class="fu">c</span>(<span class="st">"E2"</span>, <span class="st">"KO"</span>), <span class="at">log10.ind=</span><span class="cn">TRUE</span>)</span>
<span id="cb10-32"><a href="#cb10-32" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb10-33"><a href="#cb10-33" aria-hidden="true" tabindex="-1"></a>E2_KO_heatmap <span class="ot"><-</span> <span class="fu">RRHO2_heatmap</span>(E2_KO_rrho, <span class="at">colorGradient =</span> <span class="fu">wes_palette</span>(<span class="st">"Zissou1"</span>, <span class="dv">100</span>, <span class="at">type =</span> <span class="st">"continuous"</span>), <span class="at">maximum =</span> <span class="dv">350</span>)</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
</details>
<div class="cell-output-display">
<div class="quarto-figure quarto-figure-center">
<figure class="figure">
<p><img src="manuscript_figures_files/figure-html/figure_2d-1.png" class="img-fluid quarto-figure quarto-figure-center figure-img" width="672"></p>
</figure>
</div>
</div>
</div>
<p><strong>Figure 2d) Rank-Rank Hypergeometric Overlap (RRHO) heatmap comparing expression signatures between <em>APOE</em>-KO vs <em>APOE3</em> and <em>APOE2</em> vs <em>APOE3</em>.</strong></p>
<div class="cell" data-layout-align="center">
<details class="code-fold">
<summary>Code</summary>
<div class="sourceCode cell-code" id="cb11"><pre class="sourceCode r code-with-copy"><code class="sourceCode r"><span id="cb11-1"><a href="#cb11-1" aria-hidden="true" tabindex="-1"></a><span class="do">## Figure 2e: RRHO E4 and KO heatmap ##</span></span>
<span id="cb11-2"><a href="#cb11-2" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb11-3"><a href="#cb11-3" aria-hidden="true" tabindex="-1"></a><span class="co"># Set rrho p-value for E4 vs E3</span></span>
<span id="cb11-4"><a href="#cb11-4" aria-hidden="true" tabindex="-1"></a>E4vsE3_rrho <span class="ot"><-</span> E4vsE3_rna[, <span class="fu">c</span>(<span class="st">"entrezgene_id"</span>, <span class="st">"pvalue"</span>, <span class="st">"log2FoldChange"</span>)]</span>
<span id="cb11-5"><a href="#cb11-5" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb11-6"><a href="#cb11-6" aria-hidden="true" tabindex="-1"></a>E4vsE3_rrho<span class="sc">$</span>rrho_pvalue <span class="ot"><-</span> <span class="dv">0</span></span>
<span id="cb11-7"><a href="#cb11-7" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb11-8"><a href="#cb11-8" aria-hidden="true" tabindex="-1"></a>up_indices <span class="ot"><-</span> E4vsE3_rrho<span class="sc">$</span>log2FoldChange <span class="sc">></span> <span class="dv">0</span></span>
<span id="cb11-9"><a href="#cb11-9" aria-hidden="true" tabindex="-1"></a>down_indices <span class="ot"><-</span> E4vsE3_rrho<span class="sc">$</span>log2FoldChange <span class="sc"><</span> <span class="dv">0</span></span>
<span id="cb11-10"><a href="#cb11-10" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb11-11"><a href="#cb11-11" aria-hidden="true" tabindex="-1"></a>E4vsE3_rrho<span class="sc">$</span>rrho_pvalue[up_indices] <span class="ot"><-</span> <span class="sc">-</span><span class="fu">log10</span>(E4vsE3_rrho<span class="sc">$</span>pvalue[up_indices]) <span class="sc">*</span> E4vsE3_rrho<span class="sc">$</span>log2FoldChange[up_indices]</span>
<span id="cb11-12"><a href="#cb11-12" aria-hidden="true" tabindex="-1"></a>E4vsE3_rrho<span class="sc">$</span>rrho_pvalue[down_indices] <span class="ot"><-</span> <span class="sc">-</span><span class="fu">log10</span>(E4vsE3_rrho<span class="sc">$</span>pvalue[down_indices]) <span class="sc">*</span> E4vsE3_rrho<span class="sc">$</span>log2FoldChange[down_indices]</span>
<span id="cb11-13"><a href="#cb11-13" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb11-14"><a href="#cb11-14" aria-hidden="true" tabindex="-1"></a>E4vsE3_rrho <span class="ot"><-</span> E4vsE3_rrho[,<span class="fu">c</span>(<span class="st">"entrezgene_id"</span>, <span class="st">"rrho_pvalue"</span>)]</span>
<span id="cb11-15"><a href="#cb11-15" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb11-16"><a href="#cb11-16" aria-hidden="true" tabindex="-1"></a>E4_KO_rrho <span class="ot"><-</span> <span class="fu">RRHO2_initialize</span>(<span class="fu">na.omit</span>(E4vsE3_rrho), <span class="fu">na.omit</span>(KOvsE3_rrho), <span class="at">labels =</span> <span class="fu">c</span>(<span class="st">"E4"</span>, <span class="st">"KO"</span>), <span class="at">log10.ind=</span><span class="cn">TRUE</span>)</span>
<span id="cb11-17"><a href="#cb11-17" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb11-18"><a href="#cb11-18" aria-hidden="true" tabindex="-1"></a>E4_KO_heatmap <span class="ot"><-</span> <span class="fu">RRHO2_heatmap</span>(E4_KO_rrho, <span class="at">colorGradient =</span> <span class="fu">wes_palette</span>(<span class="st">"Zissou1"</span>, <span class="dv">100</span>, <span class="at">type =</span> <span class="st">"continuous"</span>), <span class="at">maximum =</span> <span class="dv">350</span>)</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
</details>
<div class="cell-output-display">
<div class="quarto-figure quarto-figure-center">
<figure class="figure">
<p><img src="manuscript_figures_files/figure-html/figure_2e-1.png" class="img-fluid quarto-figure quarto-figure-center figure-img" width="672"></p>
</figure>
</div>
</div>
</div>
<p><strong>Figure 2e) Rank-Rank Hypergeometric Overlap (RRHO) heatmap comparing expression signatures between <em>APOE</em>-KO vs <em>APOE3</em> and <em>APOE4</em> vs <em>APOE3</em>.</strong></p>
<div class="cell" data-layout-align="center">
<details class="code-fold">
<summary>Code</summary>
<div class="sourceCode cell-code" id="cb12"><pre class="sourceCode r code-with-copy"><code class="sourceCode r"><span id="cb12-1"><a href="#cb12-1" aria-hidden="true" tabindex="-1"></a>E4_uu <span class="ot"><-</span> <span class="fu">data.frame</span>(<span class="at">gene=</span>E4_KO_rrho[[<span class="st">"genelist_uu"</span>]][[<span class="st">"gene_list_overlap_uu"</span>]])</span>
<span id="cb12-2"><a href="#cb12-2" aria-hidden="true" tabindex="-1"></a>E4_uu<span class="sc">$</span>direction <span class="ot"><-</span> <span class="st">"E4_uu"</span></span>
<span id="cb12-3"><a href="#cb12-3" aria-hidden="true" tabindex="-1"></a>E4_uu<span class="sc">$</span>genotype <span class="ot"><-</span> <span class="st">"E4"</span></span>
<span id="cb12-4"><a href="#cb12-4" aria-hidden="true" tabindex="-1"></a>E4_uu <span class="ot"><-</span> <span class="fu">merge</span>(E4_uu, E4vsE3_rna, <span class="at">by.x=</span><span class="st">"gene"</span>, <span class="at">by.y=</span><span class="st">"entrezgene_id"</span>)</span>
<span id="cb12-5"><a href="#cb12-5" aria-hidden="true" tabindex="-1"></a><span class="fu">names</span>(E4_uu) <span class="ot"><-</span> <span class="fu">gsub</span>(<span class="st">"log2FoldChange"</span>, <span class="st">"log2FoldChange_E4"</span>, <span class="fu">names</span>(E4_uu))</span>
<span id="cb12-6"><a href="#cb12-6" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb12-7"><a href="#cb12-7" aria-hidden="true" tabindex="-1"></a>E2_uu <span class="ot"><-</span> <span class="fu">data.frame</span>(<span class="at">gene=</span>E2_KO_rrho[[<span class="st">"genelist_uu"</span>]][[<span class="st">"gene_list_overlap_uu"</span>]])</span>
<span id="cb12-8"><a href="#cb12-8" aria-hidden="true" tabindex="-1"></a>E2_uu<span class="sc">$</span>direction <span class="ot"><-</span> <span class="st">"E2_uu"</span></span>
<span id="cb12-9"><a href="#cb12-9" aria-hidden="true" tabindex="-1"></a>E2_uu<span class="sc">$</span>genotype <span class="ot"><-</span> <span class="st">"E2"</span></span>
<span id="cb12-10"><a href="#cb12-10" aria-hidden="true" tabindex="-1"></a>E2_uu <span class="ot"><-</span> <span class="fu">merge</span>(E2_uu, E2vsE3_rna, <span class="at">by.x=</span><span class="st">"gene"</span>, <span class="at">by.y=</span><span class="st">"entrezgene_id"</span>)</span>
<span id="cb12-11"><a href="#cb12-11" aria-hidden="true" tabindex="-1"></a><span class="fu">names</span>(E2_uu) <span class="ot"><-</span> <span class="fu">gsub</span>(<span class="st">"log2FoldChange"</span>, <span class="st">"log2FoldChange_E2"</span>, <span class="fu">names</span>(E2_uu))</span>
<span id="cb12-12"><a href="#cb12-12" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb12-13"><a href="#cb12-13" aria-hidden="true" tabindex="-1"></a>E4_dd <span class="ot"><-</span> <span class="fu">data.frame</span>(<span class="at">gene=</span>E4_KO_rrho[[<span class="st">"genelist_dd"</span>]][[<span class="st">"gene_list_overlap_dd"</span>]])</span>
<span id="cb12-14"><a href="#cb12-14" aria-hidden="true" tabindex="-1"></a>E4_dd<span class="sc">$</span>direction <span class="ot"><-</span> <span class="st">"E4_dd"</span></span>
<span id="cb12-15"><a href="#cb12-15" aria-hidden="true" tabindex="-1"></a>E4_dd<span class="sc">$</span>genotype <span class="ot"><-</span> <span class="st">"E4"</span></span>
<span id="cb12-16"><a href="#cb12-16" aria-hidden="true" tabindex="-1"></a>E4_dd <span class="ot"><-</span> <span class="fu">merge</span>(E4_dd, E4vsE3_rna, <span class="at">by.x=</span><span class="st">"gene"</span>, <span class="at">by.y=</span><span class="st">"entrezgene_id"</span>)</span>
<span id="cb12-17"><a href="#cb12-17" aria-hidden="true" tabindex="-1"></a><span class="fu">names</span>(E4_dd) <span class="ot"><-</span> <span class="fu">gsub</span>(<span class="st">"log2FoldChange"</span>, <span class="st">"log2FoldChange_E4"</span>, <span class="fu">names</span>(E4_dd))</span>
<span id="cb12-18"><a href="#cb12-18" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb12-19"><a href="#cb12-19" aria-hidden="true" tabindex="-1"></a>E2_dd <span class="ot"><-</span> <span class="fu">data.frame</span>(<span class="at">gene=</span>E2_KO_rrho[[<span class="st">"genelist_dd"</span>]][[<span class="st">"gene_list_overlap_dd"</span>]])</span>
<span id="cb12-20"><a href="#cb12-20" aria-hidden="true" tabindex="-1"></a>E2_dd<span class="sc">$</span>direction <span class="ot"><-</span> <span class="st">"E2_dd"</span></span>
<span id="cb12-21"><a href="#cb12-21" aria-hidden="true" tabindex="-1"></a>E2_dd<span class="sc">$</span>genotype <span class="ot"><-</span> <span class="st">"E2"</span></span>
<span id="cb12-22"><a href="#cb12-22" aria-hidden="true" tabindex="-1"></a>E2_dd <span class="ot"><-</span> <span class="fu">merge</span>(E2_dd, E2vsE3_rna, <span class="at">by.x=</span><span class="st">"gene"</span>, <span class="at">by.y=</span><span class="st">"entrezgene_id"</span>)</span>
<span id="cb12-23"><a href="#cb12-23" aria-hidden="true" tabindex="-1"></a><span class="fu">names</span>(E2_dd) <span class="ot"><-</span> <span class="fu">gsub</span>(<span class="st">"log2FoldChange"</span>, <span class="st">"log2FoldChange_E2"</span>, <span class="fu">names</span>(E2_dd))</span>
<span id="cb12-24"><a href="#cb12-24" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb12-25"><a href="#cb12-25" aria-hidden="true" tabindex="-1"></a><span class="co"># get genes which are shared across the comparisons </span></span>
<span id="cb12-26"><a href="#cb12-26" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb12-27"><a href="#cb12-27" aria-hidden="true" tabindex="-1"></a>uu <span class="ot"><-</span> <span class="fu">merge</span>(E2_uu, E4_uu, <span class="at">by=</span><span class="st">"gene"</span>)</span>
<span id="cb12-28"><a href="#cb12-28" aria-hidden="true" tabindex="-1"></a>dd <span class="ot"><-</span> <span class="fu">merge</span>(E2_dd, E4_dd, <span class="at">by=</span><span class="st">"gene"</span>)</span>
<span id="cb12-29"><a href="#cb12-29" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb12-30"><a href="#cb12-30" aria-hidden="true" tabindex="-1"></a>KO_overlap <span class="ot"><-</span> <span class="fu">rbind</span>(uu, dd)</span>
<span id="cb12-31"><a href="#cb12-31" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb12-32"><a href="#cb12-32" aria-hidden="true" tabindex="-1"></a>KO_overlap<span class="sc">$</span>Significant <span class="ot"><-</span> <span class="st">"Not significant"</span></span>
<span id="cb12-33"><a href="#cb12-33" aria-hidden="true" tabindex="-1"></a>KO_overlap<span class="sc">$</span>Significant[KO_overlap<span class="sc">$</span>fdr.x<span class="sc"><</span><span class="fl">0.05</span>] <span class="ot"><-</span> <span class="st">"FDR < 0.05 in APOE2"</span></span>
<span id="cb12-34"><a href="#cb12-34" aria-hidden="true" tabindex="-1"></a>KO_overlap<span class="sc">$</span>Significant[KO_overlap<span class="sc">$</span>fdr.y<span class="sc"><</span><span class="fl">0.05</span>] <span class="ot"><-</span> <span class="st">"FDR < 0.05 in APOE4"</span></span>
<span id="cb12-35"><a href="#cb12-35" aria-hidden="true" tabindex="-1"></a>KO_overlap<span class="sc">$</span>Significant[KO_overlap<span class="sc">$</span>fdr.x<span class="sc"><</span><span class="fl">0.05</span> <span class="sc">&</span> KO_overlap<span class="sc">$</span>fdr.y <span class="sc"><</span> <span class="fl">0.05</span>] <span class="ot"><-</span> <span class="st">"FDR < 0.05 in both"</span></span>
<span id="cb12-36"><a href="#cb12-36" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb12-37"><a href="#cb12-37" aria-hidden="true" tabindex="-1"></a><span class="fu">ggplot</span>(KO_overlap, <span class="fu">aes</span>(log2FoldChange_E2, log2FoldChange_E4, <span class="at">colour=</span>Significant)) <span class="sc">+</span> <span class="fu">geom_point</span>(<span class="at">size=</span><span class="dv">1</span>) <span class="sc">+</span> </span>
<span id="cb12-38"><a href="#cb12-38" aria-hidden="true" tabindex="-1"></a> <span class="fu">xlab</span>(<span class="st">"logFC of genes with similar expression profiles across </span><span class="sc">\n</span><span class="st"> APOE2 vs APOE3 and APOE-KO vs APOE3"</span>) <span class="sc">+</span> </span>
<span id="cb12-39"><a href="#cb12-39" aria-hidden="true" tabindex="-1"></a> <span class="fu">ylab</span>(<span class="st">"logFC of genes with similar expression profiles across </span><span class="sc">\n</span><span class="st"> APOE4 vs APOE3 and APOE-KO vs APOE3"</span>) <span class="sc">+</span> </span>
<span id="cb12-40"><a href="#cb12-40" aria-hidden="true" tabindex="-1"></a> <span class="fu">theme_classic</span>() <span class="sc">+</span> <span class="fu">ylim</span>(<span class="sc">-</span><span class="dv">8</span>,<span class="dv">6</span>) <span class="sc">+</span> <span class="fu">xlim</span>(<span class="sc">-</span><span class="dv">8</span>,<span class="dv">6</span>) <span class="sc">+</span> </span>
<span id="cb12-41"><a href="#cb12-41" aria-hidden="true" tabindex="-1"></a> <span class="fu">scale_colour_manual</span>(<span class="at">values=</span><span class="fu">c</span>(<span class="st">"#DCCA2CFF"</span>, <span class="st">"#92BBD9FF"</span>, <span class="st">"peru"</span>, <span class="st">"moccasin"</span>)) <span class="sc">+</span> </span>
<span id="cb12-42"><a href="#cb12-42" aria-hidden="true" tabindex="-1"></a> <span class="fu">theme</span>(<span class="at">text =</span> <span class="fu">element_text</span>(<span class="at">size=</span><span class="dv">14</span>)) <span class="sc">+</span></span>
<span id="cb12-43"><a href="#cb12-43" aria-hidden="true" tabindex="-1"></a> <span class="fu">geom_vline</span>(<span class="at">xintercept=</span><span class="dv">0</span>, <span class="at">linetype=</span><span class="st">"dashed"</span>, <span class="at">color=</span><span class="st">"darkgrey"</span>) <span class="sc">+</span> </span>
<span id="cb12-44"><a href="#cb12-44" aria-hidden="true" tabindex="-1"></a> <span class="fu">geom_hline</span>(<span class="at">yintercept=</span><span class="dv">0</span>, <span class="at">linetype=</span><span class="st">"dashed"</span>, <span class="at">color=</span><span class="st">"darkgrey"</span>)</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
</details>
<div class="cell-output-display">
<div class="quarto-figure quarto-figure-center">
<figure class="figure">
<p><img src="manuscript_figures_files/figure-html/figure_2f-1.png" class="img-fluid quarto-figure quarto-figure-center figure-img" width="768"></p>
</figure>
</div>
</div>
</div>
<div class="cell" data-layout-align="center">
<details class="code-fold">
<summary>Code</summary>
<div class="sourceCode cell-code" id="cb13"><pre class="sourceCode r code-with-copy"><code class="sourceCode r"><span id="cb13-1"><a href="#cb13-1" aria-hidden="true" tabindex="-1"></a><span class="do">## Figure 2f: MAGMA gene set analysis with AD GWAS ##</span></span>
<span id="cb13-2"><a href="#cb13-2" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb13-3"><a href="#cb13-3" aria-hidden="true" tabindex="-1"></a>magma_res <span class="ot"><-</span> <span class="fu">read.csv</span>(<span class="st">"data/MAGMA_DEGs_AD_GWAS_res.csv"</span>)</span>
<span id="cb13-4"><a href="#cb13-4" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb13-5"><a href="#cb13-5" aria-hidden="true" tabindex="-1"></a>magma_res<span class="sc">$</span>VARIABLE <span class="ot"><-</span> <span class="fu">factor</span>(magma_res<span class="sc">$</span>VARIABLE, <span class="at">levels=</span><span class="fu">c</span>(<span class="st">"Up in E2vsE3"</span>, <span class="st">"Down in E2vsE3"</span>, <span class="st">"Up in E4vsE3"</span>, <span class="st">"Down in E4vsE3"</span>, <span class="st">"Up in E4vsE2"</span>, <span class="st">"Down in E4vsE2"</span>, <span class="st">"Up in KOvsE3"</span>, <span class="st">"Down in KOvsE3"</span>))</span>
<span id="cb13-6"><a href="#cb13-6" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb13-7"><a href="#cb13-7" aria-hidden="true" tabindex="-1"></a>magma_plot <span class="ot"><-</span> <span class="fu">ggplot</span>(magma_res, <span class="fu">aes</span>(<span class="at">x=</span><span class="sc">-</span><span class="fu">log10</span>(fdr), <span class="at">y=</span>VARIABLE, <span class="at">fill=</span>VARIABLE)) <span class="sc">+</span> </span>
<span id="cb13-8"><a href="#cb13-8" aria-hidden="true" tabindex="-1"></a> <span class="fu">geom_col</span>(<span class="at">colour=</span><span class="st">"black"</span>) <span class="sc">+</span> <span class="fu">theme_bw</span>() <span class="sc">+</span> <span class="fu">xlab</span>(<span class="st">"-log10(FDR)"</span>) <span class="sc">+</span> <span class="fu">ylab</span>(<span class="st">"DEGs"</span>) <span class="sc">+</span> </span>
<span id="cb13-9"><a href="#cb13-9" aria-hidden="true" tabindex="-1"></a> <span class="fu">theme</span>(<span class="at">text =</span> <span class="fu">element_text</span>(<span class="at">size=</span><span class="dv">14</span>)) <span class="sc">+</span> </span>
<span id="cb13-10"><a href="#cb13-10" aria-hidden="true" tabindex="-1"></a> <span class="fu">theme</span>(<span class="at">legend.position =</span> <span class="st">"none"</span>, <span class="at">plot.title =</span> <span class="fu">element_text</span>(<span class="at">size =</span> <span class="dv">14</span>, <span class="at">hjust=</span><span class="fl">0.5</span>)) <span class="sc">+</span> </span>
<span id="cb13-11"><a href="#cb13-11" aria-hidden="true" tabindex="-1"></a> <span class="fu">geom_vline</span>(<span class="at">xintercept=</span><span class="sc">-</span><span class="fu">log10</span>(<span class="fl">0.05</span>), <span class="at">linetype=</span><span class="st">'dashed'</span>, <span class="at">col =</span> <span class="st">'slategrey'</span>) <span class="sc">+</span> </span>
<span id="cb13-12"><a href="#cb13-12" aria-hidden="true" tabindex="-1"></a> <span class="fu">ggtitle</span>(<span class="st">"MAGMA gene set analysis using AD GWAS"</span>) <span class="sc">+</span> <span class="fu">facet_wrap</span>(<span class="sc">~</span>GWAS) <span class="sc">+</span> </span>
<span id="cb13-13"><a href="#cb13-13" aria-hidden="true" tabindex="-1"></a> <span class="fu">scale_y_discrete</span>(<span class="at">limits=</span>rev) <span class="sc">+</span> </span>
<span id="cb13-14"><a href="#cb13-14" aria-hidden="true" tabindex="-1"></a> <span class="fu">scale_fill_manual</span>(<span class="at">values =</span> <span class="fu">c</span>(<span class="st">"#C3DAEAFF"</span>, <span class="st">"#92BBD9FF"</span>, <span class="st">"#ECE28BFF"</span>, <span class="st">"#DCCA2CFF"</span>, <span class="st">"#B1D5BBFF"</span>, <span class="st">"#6FB382FF"</span>, <span class="st">"#9DAFC3FF"</span>, <span class="st">"#4D6D93FF"</span>))</span>
<span id="cb13-15"><a href="#cb13-15" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb13-16"><a href="#cb13-16" aria-hidden="true" tabindex="-1"></a><span class="fu">plot</span>(magma_plot)</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
</details>
<div class="cell-output-display">
<div class="quarto-figure quarto-figure-center">
<figure class="figure">
<p><img src="manuscript_figures_files/figure-html/figure_2g-1.png" class="img-fluid quarto-figure quarto-figure-center figure-img" width="672"></p>
</figure>
</div>
</div>
</div>
<p><strong>Figure 2g) MAGMA gene set analysis using the differentially expressed genes across the <em>APOE</em> groups with three independent AD GWAS.</strong></p>
<p><strong>Figure 3: Differentially expressed genes exhibit differential chromatin accessibility in their vicinity.</strong></p>
<div class="cell" data-layout-align="center">
<details class="code-fold">
<summary>Code</summary>
<div class="sourceCode cell-code" id="cb14"><pre class="sourceCode r code-with-copy"><code class="sourceCode r"><span id="cb14-1"><a href="#cb14-1" aria-hidden="true" tabindex="-1"></a><span class="do">## Figure 3a: E2vsE3 DAR heatmap with RNA-seq genes ## </span></span>
<span id="cb14-2"><a href="#cb14-2" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb14-3"><a href="#cb14-3" aria-hidden="true" tabindex="-1"></a><span class="co"># normalise counts </span></span>
<span id="cb14-4"><a href="#cb14-4" aria-hidden="true" tabindex="-1"></a>counts_norm <span class="ot"><-</span> <span class="fu">normTransform</span>(atac_dds)</span>
<span id="cb14-5"><a href="#cb14-5" aria-hidden="true" tabindex="-1"></a>counts_norm_assay <span class="ot"><-</span> <span class="fu">assay</span>(counts_norm)</span>
<span id="cb14-6"><a href="#cb14-6" aria-hidden="true" tabindex="-1"></a>counts_z <span class="ot"><-</span> <span class="fu">t</span>(<span class="fu">scale</span>(<span class="fu">t</span>(counts_norm_assay)))</span>
<span id="cb14-7"><a href="#cb14-7" aria-hidden="true" tabindex="-1"></a>counts_z <span class="ot"><-</span> counts_z[,<span class="fu">c</span>(<span class="st">"E2_1"</span>, <span class="st">"E2_2"</span>, <span class="st">"E2_3"</span>, <span class="st">"E2_4"</span>, <span class="st">"E2_5"</span>, <span class="st">"E3_1"</span>, <span class="st">"E3_2"</span>, <span class="st">"E3_3"</span>, <span class="st">"E3_4"</span>, <span class="st">"E3_5"</span>, <span class="st">"E4_1"</span>, <span class="st">"E4_2"</span>, <span class="st">"E4_3"</span>, <span class="st">"E4_5"</span>, <span class="st">"KO_3"</span>, <span class="st">"KO_5"</span>)]</span>
<span id="cb14-8"><a href="#cb14-8" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb14-9"><a href="#cb14-9" aria-hidden="true" tabindex="-1"></a>indices <span class="ot"><-</span> <span class="fu">match</span>(E2vsE3_atac_sig<span class="sc">$</span>Peak, <span class="fu">rownames</span>(counts_z))</span>
<span id="cb14-10"><a href="#cb14-10" aria-hidden="true" tabindex="-1"></a>E2vsE3_counts <span class="ot"><-</span> counts_z[indices, ]</span>
<span id="cb14-11"><a href="#cb14-11" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb14-12"><a href="#cb14-12" aria-hidden="true" tabindex="-1"></a>indices <span class="ot"><-</span> <span class="fu">match</span>(E4vsE3_atac_sig<span class="sc">$</span>Peak, <span class="fu">rownames</span>(counts_z))</span>
<span id="cb14-13"><a href="#cb14-13" aria-hidden="true" tabindex="-1"></a>E4vsE3_counts <span class="ot"><-</span> counts_z[indices, ]</span>
<span id="cb14-14"><a href="#cb14-14" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb14-15"><a href="#cb14-15" aria-hidden="true" tabindex="-1"></a>indices <span class="ot"><-</span> <span class="fu">match</span>(KOvsE3_atac_sig<span class="sc">$</span>Peak, <span class="fu">rownames</span>(counts_z))</span>
<span id="cb14-16"><a href="#cb14-16" aria-hidden="true" tabindex="-1"></a>KOvsE3_counts <span class="ot"><-</span> counts_z[indices, ]</span>
<span id="cb14-17"><a href="#cb14-17" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb14-18"><a href="#cb14-18" aria-hidden="true" tabindex="-1"></a>indices <span class="ot"><-</span> <span class="fu">match</span>(E4vsE2_atac_sig<span class="sc">$</span>Peak, <span class="fu">rownames</span>(counts_z))</span>
<span id="cb14-19"><a href="#cb14-19" aria-hidden="true" tabindex="-1"></a>E4vsE2_counts <span class="ot"><-</span> counts_z[indices, ]</span>
<span id="cb14-20"><a href="#cb14-20" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb14-21"><a href="#cb14-21" aria-hidden="true" tabindex="-1"></a>mat <span class="ot"><-</span> E2vsE3_counts</span>
<span id="cb14-22"><a href="#cb14-22" aria-hidden="true" tabindex="-1"></a><span class="fu">rownames</span>(mat) <span class="ot"><-</span> E2vsE3_atac_genes<span class="sc">$</span>SYMBOL</span>
<span id="cb14-23"><a href="#cb14-23" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb14-24"><a href="#cb14-24" aria-hidden="true" tabindex="-1"></a><span class="co"># overlapping genes between atac-seq and rna-seq </span></span>
<span id="cb14-25"><a href="#cb14-25" aria-hidden="true" tabindex="-1"></a>overlap_genes <span class="ot"><-</span> <span class="fu">rownames</span>(mat)[E2vsE3_atac_genes<span class="sc">$</span>SYMBOL <span class="sc">%in%</span> E2vsE3_rna_genes<span class="sc">$</span>hgnc_symbol]</span>
<span id="cb14-26"><a href="#cb14-26" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb14-27"><a href="#cb14-27" aria-hidden="true" tabindex="-1"></a><span class="co"># function to make selected rownames bold</span></span>
<span id="cb14-28"><a href="#cb14-28" aria-hidden="true" tabindex="-1"></a>make_bold_names <span class="ot"><-</span> <span class="cf">function</span>(mat, rc_fun, rc_names) {</span>
<span id="cb14-29"><a href="#cb14-29" aria-hidden="true" tabindex="-1"></a> bold_names <span class="ot"><-</span> <span class="fu">rc_fun</span>(mat)[<span class="dv">1</span><span class="sc">:</span><span class="dv">20</span>]</span>
<span id="cb14-30"><a href="#cb14-30" aria-hidden="true" tabindex="-1"></a> ids <span class="ot"><-</span> rc_names <span class="sc">%>%</span> <span class="fu">match</span>(bold_names)</span>
<span id="cb14-31"><a href="#cb14-31" aria-hidden="true" tabindex="-1"></a> ids <span class="sc">%>%</span></span>
<span id="cb14-32"><a href="#cb14-32" aria-hidden="true" tabindex="-1"></a> purrr<span class="sc">::</span><span class="fu">walk</span>(</span>
<span id="cb14-33"><a href="#cb14-33" aria-hidden="true" tabindex="-1"></a> <span class="cf">function</span>(i)</span>
<span id="cb14-34"><a href="#cb14-34" aria-hidden="true" tabindex="-1"></a> bold_names[i] <span class="ot"><<-</span></span>
<span id="cb14-35"><a href="#cb14-35" aria-hidden="true" tabindex="-1"></a> <span class="fu">bquote</span>(<span class="fu">bold</span>(.(<span class="fu">rc_fun</span>(mat)[i]))) <span class="sc">%>%</span></span>
<span id="cb14-36"><a href="#cb14-36" aria-hidden="true" tabindex="-1"></a> <span class="fu">as.expression</span>()</span>
<span id="cb14-37"><a href="#cb14-37" aria-hidden="true" tabindex="-1"></a> )</span>
<span id="cb14-38"><a href="#cb14-38" aria-hidden="true" tabindex="-1"></a> bold_names</span>
<span id="cb14-39"><a href="#cb14-39" aria-hidden="true" tabindex="-1"></a>}</span>
<span id="cb14-40"><a href="#cb14-40" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb14-41"><a href="#cb14-41" aria-hidden="true" tabindex="-1"></a>mat_df <span class="ot"><-</span> <span class="fu">data.frame</span>(mat)</span>
<span id="cb14-42"><a href="#cb14-42" aria-hidden="true" tabindex="-1"></a>mat_df<span class="sc">$</span>direction <span class="ot"><-</span> <span class="fu">ifelse</span>(mat_df<span class="sc">$</span>E2_1<span class="sc">></span><span class="dv">0</span>, <span class="st">"Up"</span>, <span class="st">"Down"</span>)</span>
<span id="cb14-43"><a href="#cb14-43" aria-hidden="true" tabindex="-1"></a>mat_df<span class="sc">$</span>label_colour<span class="ot">=</span><span class="fu">ifelse</span>(mat_df<span class="sc">$</span>direction<span class="sc">==</span><span class="st">"Up"</span>,<span class="st">"#B2182B"</span>,<span class="st">"#4D4D4D"</span>)</span>
<span id="cb14-44"><a href="#cb14-44" aria-hidden="true" tabindex="-1"></a><span class="co"># group replicates </span></span>
<span id="cb14-45"><a href="#cb14-45" aria-hidden="true" tabindex="-1"></a>group_col <span class="ot"><-</span> <span class="fu">data.frame</span>(<span class="at">Group =</span> <span class="fu">c</span>(<span class="fu">rep</span>(<span class="st">"E2"</span>,<span class="dv">5</span>), <span class="fu">rep</span>(<span class="st">"E3"</span>,<span class="dv">5</span>), <span class="fu">rep</span>(<span class="st">"E4"</span>, <span class="dv">4</span>), <span class="fu">rep</span>(<span class="st">"KO"</span>,<span class="dv">2</span>)))</span>
<span id="cb14-46"><a href="#cb14-46" aria-hidden="true" tabindex="-1"></a><span class="fu">rownames</span>(group_col) <span class="ot"><-</span> <span class="fu">colnames</span>(mat)</span>
<span id="cb14-47"><a href="#cb14-47" aria-hidden="true" tabindex="-1"></a>group_colour <span class="ot">=</span> <span class="fu">list</span>(<span class="at">Group =</span> <span class="fu">c</span>(<span class="at">E2 =</span> <span class="st">"#92BBD9FF"</span>, </span>
<span id="cb14-48"><a href="#cb14-48" aria-hidden="true" tabindex="-1"></a> <span class="at">E3 =</span> <span class="st">"#ECE28BFF"</span>,</span>
<span id="cb14-49"><a href="#cb14-49" aria-hidden="true" tabindex="-1"></a> <span class="at">E4 =</span> <span class="st">"#6FB382FF"</span>, </span>
<span id="cb14-50"><a href="#cb14-50" aria-hidden="true" tabindex="-1"></a> <span class="at">KO =</span> <span class="st">"#4D6D93FF"</span>))</span>
<span id="cb14-51"><a href="#cb14-51" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb14-52"><a href="#cb14-52" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb14-53"><a href="#cb14-53" aria-hidden="true" tabindex="-1"></a>E2vsE3_pheatmap <span class="ot"><-</span> <span class="fu">pheatmap</span>(mat, <span class="at">color =</span> <span class="fu">colorRampPalette</span>(<span class="fu">rev</span>(<span class="fu">brewer.pal</span>(<span class="at">n =</span> <span class="dv">7</span>, <span class="at">name =</span> <span class="st">"RdGy"</span>)))(<span class="dv">100</span>), </span>
<span id="cb14-54"><a href="#cb14-54" aria-hidden="true" tabindex="-1"></a> <span class="at">cluster_rows=</span><span class="cn">TRUE</span>, <span class="at">cluster_cols=</span><span class="cn">FALSE</span>, <span class="at">fontsize_col =</span> <span class="dv">16</span>, </span>
<span id="cb14-55"><a href="#cb14-55" aria-hidden="true" tabindex="-1"></a> <span class="at">fontsize_row=</span><span class="dv">12</span>, <span class="at">labels_row =</span> <span class="fu">make_bold_names</span>(mat,rownames,overlap_genes),</span>
<span id="cb14-56"><a href="#cb14-56" aria-hidden="true" tabindex="-1"></a> <span class="at">cellwidth =</span> <span class="dv">15</span>, <span class="at">cellheight=</span><span class="dv">10</span>, <span class="at">annotation_col =</span> group_col,</span>
<span id="cb14-57"><a href="#cb14-57" aria-hidden="true" tabindex="-1"></a> <span class="at">annotation_colors =</span> group_colour, <span class="at">labels_col =</span> <span class="st">""</span>, <span class="at">main =</span> <span class="st">"E2 vs E3"</span>)</span>
<span id="cb14-58"><a href="#cb14-58" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb14-59"><a href="#cb14-59" aria-hidden="true" tabindex="-1"></a>cols<span class="ot">=</span>mat_df[E2vsE3_pheatmap<span class="sc">$</span>tree_row<span class="sc">$</span>order,]<span class="sc">$</span>label_colour</span>
<span id="cb14-60"><a href="#cb14-60" aria-hidden="true" tabindex="-1"></a>E2vsE3_pheatmap<span class="sc">$</span>gtable<span class="sc">$</span>grobs[[<span class="dv">5</span>]]<span class="sc">$</span>gp<span class="ot">=</span><span class="fu">gpar</span>(<span class="at">col=</span>cols, <span class="at">fontsize =</span> <span class="dv">12</span>)</span>
<span id="cb14-61"><a href="#cb14-61" aria-hidden="true" tabindex="-1"></a>E2vsE3_pheatmap</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
</details>
<div class="cell-output-display">
<div class="quarto-figure quarto-figure-center">
<figure class="figure">
<p><img src="manuscript_figures_files/figure-html/figure_3a-1.png" class="img-fluid quarto-figure quarto-figure-center figure-img" width="480"></p>
</figure>
</div>
</div>
</div>
<p><strong>Figure 3a) Heatmap showing differential chromatin accessibility of significant peaks (FDR < 0.05) when comparing <em>APOE2</em> vs <em>APOE3</em>-expressing microglia.</strong></p>
<div class="cell" data-layout-align="center">
<details class="code-fold">
<summary>Code</summary>
<div class="sourceCode cell-code" id="cb15"><pre class="sourceCode r code-with-copy"><code class="sourceCode r"><span id="cb15-1"><a href="#cb15-1" aria-hidden="true" tabindex="-1"></a><span class="do">## Figure 3b: E4vsE3 DAR heatmap with RNA-seq genes ##</span></span>
<span id="cb15-2"><a href="#cb15-2" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb15-3"><a href="#cb15-3" aria-hidden="true" tabindex="-1"></a>mat <span class="ot"><-</span> E4vsE3_counts</span>
<span id="cb15-4"><a href="#cb15-4" aria-hidden="true" tabindex="-1"></a><span class="fu">rownames</span>(mat) <span class="ot"><-</span> E4vsE3_atac_genes<span class="sc">$</span>SYMBOL</span>
<span id="cb15-5"><a href="#cb15-5" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb15-6"><a href="#cb15-6" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb15-7"><a href="#cb15-7" aria-hidden="true" tabindex="-1"></a><span class="co"># overlapping genes between atac-seq and rna-seq </span></span>
<span id="cb15-8"><a href="#cb15-8" aria-hidden="true" tabindex="-1"></a>overlap_genes <span class="ot"><-</span> <span class="fu">rownames</span>(mat)[E4vsE3_atac_genes<span class="sc">$</span>SYMBOL <span class="sc">%in%</span> E4vsE3_rna_genes<span class="sc">$</span>hgnc_symbol]</span>
<span id="cb15-9"><a href="#cb15-9" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb15-10"><a href="#cb15-10" aria-hidden="true" tabindex="-1"></a><span class="co"># function to make selected rownames bold</span></span>
<span id="cb15-11"><a href="#cb15-11" aria-hidden="true" tabindex="-1"></a>make_bold_names <span class="ot"><-</span> <span class="cf">function</span>(mat, rc_fun, rc_names) {</span>
<span id="cb15-12"><a href="#cb15-12" aria-hidden="true" tabindex="-1"></a> bold_names <span class="ot"><-</span> <span class="fu">rc_fun</span>(mat)[<span class="dv">1</span><span class="sc">:</span><span class="dv">20</span>]</span>
<span id="cb15-13"><a href="#cb15-13" aria-hidden="true" tabindex="-1"></a> ids <span class="ot"><-</span> rc_names <span class="sc">%>%</span> <span class="fu">match</span>(bold_names)</span>
<span id="cb15-14"><a href="#cb15-14" aria-hidden="true" tabindex="-1"></a> ids <span class="sc">%>%</span></span>
<span id="cb15-15"><a href="#cb15-15" aria-hidden="true" tabindex="-1"></a> purrr<span class="sc">::</span><span class="fu">walk</span>(</span>
<span id="cb15-16"><a href="#cb15-16" aria-hidden="true" tabindex="-1"></a> <span class="cf">function</span>(i)</span>
<span id="cb15-17"><a href="#cb15-17" aria-hidden="true" tabindex="-1"></a> bold_names[i] <span class="ot"><<-</span></span>
<span id="cb15-18"><a href="#cb15-18" aria-hidden="true" tabindex="-1"></a> <span class="fu">bquote</span>(<span class="fu">bold</span>(.(<span class="fu">rc_fun</span>(mat)[i]))) <span class="sc">%>%</span></span>
<span id="cb15-19"><a href="#cb15-19" aria-hidden="true" tabindex="-1"></a> <span class="fu">as.expression</span>()</span>
<span id="cb15-20"><a href="#cb15-20" aria-hidden="true" tabindex="-1"></a> )</span>
<span id="cb15-21"><a href="#cb15-21" aria-hidden="true" tabindex="-1"></a> bold_names</span>
<span id="cb15-22"><a href="#cb15-22" aria-hidden="true" tabindex="-1"></a>}</span>
<span id="cb15-23"><a href="#cb15-23" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb15-24"><a href="#cb15-24" aria-hidden="true" tabindex="-1"></a>mat_df <span class="ot"><-</span> <span class="fu">data.frame</span>(mat)</span>
<span id="cb15-25"><a href="#cb15-25" aria-hidden="true" tabindex="-1"></a>mat_df<span class="sc">$</span>direction <span class="ot"><-</span> <span class="fu">ifelse</span>(mat_df<span class="sc">$</span>E4_1<span class="sc">></span><span class="dv">0</span>, <span class="st">"Up"</span>, <span class="st">"Down"</span>)</span>
<span id="cb15-26"><a href="#cb15-26" aria-hidden="true" tabindex="-1"></a>mat_df<span class="sc">$</span>label_colour<span class="ot">=</span><span class="fu">ifelse</span>(mat_df<span class="sc">$</span>direction<span class="sc">==</span><span class="st">"Up"</span>,<span class="st">"#B2182B"</span>,<span class="st">"#4D4D4D"</span>)</span>
<span id="cb15-27"><a href="#cb15-27" aria-hidden="true" tabindex="-1"></a><span class="co"># group replicates </span></span>
<span id="cb15-28"><a href="#cb15-28" aria-hidden="true" tabindex="-1"></a>group_col <span class="ot"><-</span> <span class="fu">data.frame</span>(<span class="at">Group =</span> <span class="fu">c</span>(<span class="fu">rep</span>(<span class="st">"E2"</span>,<span class="dv">5</span>), <span class="fu">rep</span>(<span class="st">"E3"</span>,<span class="dv">5</span>), <span class="fu">rep</span>(<span class="st">"E4"</span>, <span class="dv">4</span>), <span class="fu">rep</span>(<span class="st">"KO"</span>,<span class="dv">2</span>)))</span>
<span id="cb15-29"><a href="#cb15-29" aria-hidden="true" tabindex="-1"></a><span class="fu">rownames</span>(group_col) <span class="ot"><-</span> <span class="fu">colnames</span>(mat)</span>
<span id="cb15-30"><a href="#cb15-30" aria-hidden="true" tabindex="-1"></a>group_colour <span class="ot">=</span> <span class="fu">list</span>(<span class="at">Group =</span> <span class="fu">c</span>(<span class="at">E2 =</span> <span class="st">"#92BBD9FF"</span>, </span>
<span id="cb15-31"><a href="#cb15-31" aria-hidden="true" tabindex="-1"></a> <span class="at">E3 =</span> <span class="st">"#ECE28BFF"</span>,</span>
<span id="cb15-32"><a href="#cb15-32" aria-hidden="true" tabindex="-1"></a> <span class="at">E4 =</span> <span class="st">"#6FB382FF"</span>, </span>
<span id="cb15-33"><a href="#cb15-33" aria-hidden="true" tabindex="-1"></a> <span class="at">KO =</span> <span class="st">"#4D6D93FF"</span>))</span>
<span id="cb15-34"><a href="#cb15-34" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb15-35"><a href="#cb15-35" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb15-36"><a href="#cb15-36" aria-hidden="true" tabindex="-1"></a>E4vsE3_pheatmap <span class="ot"><-</span> <span class="fu">pheatmap</span>(mat, <span class="at">color =</span> <span class="fu">colorRampPalette</span>(<span class="fu">rev</span>(<span class="fu">brewer.pal</span>(<span class="at">n =</span> <span class="dv">7</span>, <span class="at">name =</span> <span class="st">"RdGy"</span>)))(<span class="dv">100</span>), </span>
<span id="cb15-37"><a href="#cb15-37" aria-hidden="true" tabindex="-1"></a> <span class="at">cluster_rows=</span><span class="cn">TRUE</span>, <span class="at">cluster_cols=</span><span class="cn">FALSE</span>, <span class="at">fontsize_col =</span> <span class="dv">16</span>, </span>
<span id="cb15-38"><a href="#cb15-38" aria-hidden="true" tabindex="-1"></a> <span class="at">fontsize_row=</span><span class="dv">12</span>, <span class="at">labels_row =</span> <span class="fu">make_bold_names</span>(mat, rownames, overlap_genes),</span>
<span id="cb15-39"><a href="#cb15-39" aria-hidden="true" tabindex="-1"></a> <span class="at">cellwidth =</span> <span class="dv">15</span>, <span class="at">cellheight=</span><span class="dv">10</span>, <span class="at">annotation_col =</span> group_col,</span>
<span id="cb15-40"><a href="#cb15-40" aria-hidden="true" tabindex="-1"></a> <span class="at">annotation_colors =</span> group_colour, <span class="at">labels_col =</span> <span class="st">""</span>, <span class="at">main =</span> <span class="st">"E4 vs E3"</span>)</span>
<span id="cb15-41"><a href="#cb15-41" aria-hidden="true" tabindex="-1"></a>cols<span class="ot">=</span>mat_df[E4vsE3_pheatmap<span class="sc">$</span>tree_row<span class="sc">$</span>order,]<span class="sc">$</span>label_colour</span>
<span id="cb15-42"><a href="#cb15-42" aria-hidden="true" tabindex="-1"></a>E4vsE3_pheatmap<span class="sc">$</span>gtable<span class="sc">$</span>grobs[[<span class="dv">5</span>]]<span class="sc">$</span>gp<span class="ot">=</span><span class="fu">gpar</span>(<span class="at">col=</span>cols, <span class="at">fontsize =</span> <span class="dv">12</span>)</span>
<span id="cb15-43"><a href="#cb15-43" aria-hidden="true" tabindex="-1"></a>E4vsE3_pheatmap</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
</details>
<div class="cell-output-display">
<div class="quarto-figure quarto-figure-center">
<figure class="figure">
<p><img src="manuscript_figures_files/figure-html/figure_3b-1.png" class="img-fluid quarto-figure quarto-figure-center figure-img" width="480"></p>
</figure>
</div>
</div>
</div>
<p><strong>Figure 3b) Heatmap showing differential chromatin accessibility of significant peaks (FDR < 0.05) when comparing <em>APOE4</em> vs <em>APOE3</em>-expressing microglia.</strong></p>
<div class="cell" data-layout-align="center">
<details class="code-fold">
<summary>Code</summary>
<div class="sourceCode cell-code" id="cb16"><pre class="sourceCode r code-with-copy"><code class="sourceCode r"><span id="cb16-1"><a href="#cb16-1" aria-hidden="true" tabindex="-1"></a><span class="do">## Figure 3d: CHCHD2 expression ## </span></span>
<span id="cb16-2"><a href="#cb16-2" aria-hidden="true" tabindex="-1"></a>chchd2 <span class="ot"><-</span> <span class="fu">plotCounts</span>(dds, <span class="at">gene=</span><span class="st">"51142"</span>, <span class="at">intgroup=</span><span class="st">"group"</span>, <span class="at">returnData =</span> <span class="cn">TRUE</span>)</span>
<span id="cb16-3"><a href="#cb16-3" aria-hidden="true" tabindex="-1"></a>my_comparisons <span class="ot"><-</span> <span class="fu">list</span>( <span class="fu">c</span>(<span class="st">"E2"</span>, <span class="st">"E3"</span>), <span class="fu">c</span>(<span class="st">"E2"</span>, <span class="st">"E4"</span>), <span class="fu">c</span>(<span class="st">"E2"</span>,<span class="st">"KO"</span>))</span>
<span id="cb16-4"><a href="#cb16-4" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb16-5"><a href="#cb16-5" aria-hidden="true" tabindex="-1"></a><span class="fu">ggplot</span>(chchd2, <span class="fu">aes</span>(<span class="at">x=</span>group, <span class="at">y=</span>count, <span class="at">fill=</span>group)) <span class="sc">+</span> </span>
<span id="cb16-6"><a href="#cb16-6" aria-hidden="true" tabindex="-1"></a> <span class="fu">geom_boxplot</span>() <span class="sc">+</span> </span>
<span id="cb16-7"><a href="#cb16-7" aria-hidden="true" tabindex="-1"></a> <span class="fu">theme_classic</span>() <span class="sc">+</span> </span>
<span id="cb16-8"><a href="#cb16-8" aria-hidden="true" tabindex="-1"></a> <span class="fu">scale_fill_manual</span>(<span class="at">values =</span> <span class="fu">ghibli_palette</span>(<span class="st">"YesterdayMedium"</span>, <span class="at">direction =</span> <span class="sc">-</span><span class="dv">1</span>)) <span class="sc">+</span> </span>
<span id="cb16-9"><a href="#cb16-9" aria-hidden="true" tabindex="-1"></a> <span class="fu">ylab</span>(<span class="st">"Counts"</span>) <span class="sc">+</span> </span>
<span id="cb16-10"><a href="#cb16-10" aria-hidden="true" tabindex="-1"></a> <span class="fu">ggtitle</span>(<span class="st">"CHCHD2"</span>) <span class="sc">+</span> </span>
<span id="cb16-11"><a href="#cb16-11" aria-hidden="true" tabindex="-1"></a> <span class="fu">xlab</span>(<span class="st">""</span>) <span class="sc">+</span> </span>
<span id="cb16-12"><a href="#cb16-12" aria-hidden="true" tabindex="-1"></a> <span class="fu">theme</span>(<span class="at">text =</span> <span class="fu">element_text</span>(<span class="at">size =</span> <span class="dv">20</span>), <span class="at">plot.title =</span> <span class="fu">element_text</span>(<span class="at">size=</span><span class="dv">20</span>,<span class="at">hjust =</span> <span class="fl">0.5</span>, <span class="at">face=</span><span class="fu">c</span>(<span class="st">"italic"</span>))) <span class="sc">+</span> <span class="fu">stat_compare_means</span>(<span class="at">comparisons =</span> my_comparisons, <span class="at">method=</span><span class="st">"t.test"</span>) <span class="sc">+</span></span>
<span id="cb16-13"><a href="#cb16-13" aria-hidden="true" tabindex="-1"></a> <span class="fu">geom_jitter</span>() <span class="sc">+</span> </span>
<span id="cb16-14"><a href="#cb16-14" aria-hidden="true" tabindex="-1"></a> <span class="fu">labs</span>(<span class="at">fill=</span><span class="cn">NULL</span>)</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
</details>
<div class="cell-output-display">
<div class="quarto-figure quarto-figure-center">
<figure class="figure">
<p><img src="manuscript_figures_files/figure-html/figure_3c-1.png" class="img-fluid quarto-figure quarto-figure-center figure-img" width="480"></p>
</figure>
</div>
</div>
</div>
<p><strong>Figure 3c) Boxplot of expression profiles of CHCHD2 shows reduced expression in the APOE4 and the APOE-KO microglia.</strong></p>
<div class="cell" data-layout-align="center">
<details class="code-fold">
<summary>Code</summary>
<div class="sourceCode cell-code" id="cb17"><pre class="sourceCode r code-with-copy"><code class="sourceCode r"><span id="cb17-1"><a href="#cb17-1" aria-hidden="true" tabindex="-1"></a><span class="do">## Figure 3d: chromatin accessibility around CHCHD2 ##</span></span>
<span id="cb17-2"><a href="#cb17-2" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb17-3"><a href="#cb17-3" aria-hidden="true" tabindex="-1"></a>start <span class="ot"><-</span> E4vsE2_atac_annot<span class="sc">$</span>geneStart[E4vsE2_atac_annot<span class="sc">$</span>SYMBOL<span class="sc">==</span><span class="st">"CHCHD2"</span>][<span class="dv">1</span>] <span class="sc">-</span><span class="dv">3000</span></span>
<span id="cb17-4"><a href="#cb17-4" aria-hidden="true" tabindex="-1"></a>end <span class="ot"><-</span> E4vsE2_atac_annot<span class="sc">$</span>geneEnd[E4vsE2_atac_annot<span class="sc">$</span>SYMBOL<span class="sc">==</span><span class="st">"CHCHD2"</span>][<span class="dv">1</span>] <span class="sc">+</span> <span class="dv">3000</span></span>
<span id="cb17-5"><a href="#cb17-5" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb17-6"><a href="#cb17-6" aria-hidden="true" tabindex="-1"></a><span class="co"># plot data tracks </span></span>
<span id="cb17-7"><a href="#cb17-7" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb17-8"><a href="#cb17-8" aria-hidden="true" tabindex="-1"></a>track_list <span class="ot"><-</span> <span class="fu">c</span>(<span class="at">KO =</span> <span class="st">"data/KO-1.bw"</span>,</span>
<span id="cb17-9"><a href="#cb17-9" aria-hidden="true" tabindex="-1"></a> <span class="at">E4 =</span> <span class="st">"data/E4-1.bw"</span>, </span>
<span id="cb17-10"><a href="#cb17-10" aria-hidden="true" tabindex="-1"></a> <span class="at">E3 =</span> <span class="st">"data/E3-1.bw"</span>, </span>
<span id="cb17-11"><a href="#cb17-11" aria-hidden="true" tabindex="-1"></a> <span class="at">E2 =</span> <span class="st">"data/E2-1.bw"</span>)</span>
<span id="cb17-12"><a href="#cb17-12" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb17-13"><a href="#cb17-13" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb17-14"><a href="#cb17-14" aria-hidden="true" tabindex="-1"></a>chchd2_region <span class="ot"><-</span> <span class="fu">toGRanges</span>(<span class="fu">data.frame</span>(<span class="at">chr=</span><span class="st">"chr7"</span>, <span class="at">start=</span>start, <span class="at">end=</span>end))</span>
<span id="cb17-15"><a href="#cb17-15" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb17-16"><a href="#cb17-16" aria-hidden="true" tabindex="-1"></a><span class="co"># set plotting parameters</span></span>
<span id="cb17-17"><a href="#cb17-17" aria-hidden="true" tabindex="-1"></a>pp <span class="ot"><-</span> <span class="fu">getDefaultPlotParams</span>(<span class="at">plot.type=</span><span class="dv">1</span>)</span>
<span id="cb17-18"><a href="#cb17-18" aria-hidden="true" tabindex="-1"></a>pp<span class="sc">$</span>leftmargin <span class="ot"><-</span> <span class="fl">0.15</span></span>
<span id="cb17-19"><a href="#cb17-19" aria-hidden="true" tabindex="-1"></a>pp<span class="sc">$</span>topmargin <span class="ot"><-</span> <span class="dv">15</span></span>
<span id="cb17-20"><a href="#cb17-20" aria-hidden="true" tabindex="-1"></a>pp<span class="sc">$</span>bottommargin <span class="ot"><-</span> <span class="dv">15</span></span>
<span id="cb17-21"><a href="#cb17-21" aria-hidden="true" tabindex="-1"></a>pp<span class="sc">$</span>ideogramheight <span class="ot"><-</span> <span class="dv">5</span></span>
<span id="cb17-22"><a href="#cb17-22" aria-hidden="true" tabindex="-1"></a>pp<span class="sc">$</span>data1inmargin <span class="ot"><-</span> <span class="dv">10</span></span>
<span id="cb17-23"><a href="#cb17-23" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb17-24"><a href="#cb17-24" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb17-25"><a href="#cb17-25" aria-hidden="true" tabindex="-1"></a>kp <span class="ot"><-</span> <span class="fu">plotKaryotype</span>(<span class="at">zoom =</span> chchd2_region, <span class="at">cex=</span><span class="fl">1.7</span>, <span class="at">plot.params =</span> pp)</span>
<span id="cb17-26"><a href="#cb17-26" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb17-27"><a href="#cb17-27" aria-hidden="true" tabindex="-1"></a><span class="fu">kpAddBaseNumbers</span>(kp, <span class="at">tick.dist =</span> <span class="dv">3000</span>, <span class="at">minor.tick.dist =</span> <span class="dv">1500</span>,</span>
<span id="cb17-28"><a href="#cb17-28" aria-hidden="true" tabindex="-1"></a> <span class="at">add.units =</span> <span class="cn">TRUE</span>, <span class="at">cex=</span><span class="fl">1.5</span>, <span class="at">digits =</span> <span class="dv">6</span>)</span>
<span id="cb17-29"><a href="#cb17-29" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb17-30"><a href="#cb17-30" aria-hidden="true" tabindex="-1"></a>chchd2_data <span class="ot"><-</span> <span class="fu">makeGenesDataFromTxDb</span>(TxDb.Hsapiens.UCSC.hg38.knownGene,</span>
<span id="cb17-31"><a href="#cb17-31" aria-hidden="true" tabindex="-1"></a> <span class="at">karyoplot=</span>kp,</span>
<span id="cb17-32"><a href="#cb17-32" aria-hidden="true" tabindex="-1"></a> <span class="at">plot.transcripts =</span> <span class="cn">TRUE</span>, </span>
<span id="cb17-33"><a href="#cb17-33" aria-hidden="true" tabindex="-1"></a> <span class="at">plot.transcripts.structure =</span> <span class="cn">TRUE</span>)</span>
<span id="cb17-34"><a href="#cb17-34" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb17-35"><a href="#cb17-35" aria-hidden="true" tabindex="-1"></a>chchd2_data<span class="sc">$</span>genes<span class="sc">$</span>name <span class="ot"><-</span> <span class="st">"CHCHD2"</span></span>
<span id="cb17-36"><a href="#cb17-36" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb17-37"><a href="#cb17-37" aria-hidden="true" tabindex="-1"></a><span class="co"># KO</span></span>
<span id="cb17-38"><a href="#cb17-38" aria-hidden="true" tabindex="-1"></a>bigwig.file <span class="ot"><-</span> track_list[[<span class="st">"KO"</span>]]</span>
<span id="cb17-39"><a href="#cb17-39" aria-hidden="true" tabindex="-1"></a>track_position <span class="ot">=</span> <span class="dv">1</span></span>
<span id="cb17-40"><a href="#cb17-40" aria-hidden="true" tabindex="-1"></a>track_colour <span class="ot">=</span> <span class="st">"#4D6D93FF"</span></span>
<span id="cb17-41"><a href="#cb17-41" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb17-42"><a href="#cb17-42" aria-hidden="true" tabindex="-1"></a><span class="co"># Adjusting the margin for tracks</span></span>
<span id="cb17-43"><a href="#cb17-43" aria-hidden="true" tabindex="-1"></a>track_margin <span class="ot"><-</span> <span class="fl">0.1</span></span>
<span id="cb17-44"><a href="#cb17-44" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb17-45"><a href="#cb17-45" aria-hidden="true" tabindex="-1"></a><span class="co"># Autotrack for genes</span></span>
<span id="cb17-46"><a href="#cb17-46" aria-hidden="true" tabindex="-1"></a>genes_track_at <span class="ot"><-</span> <span class="fu">autotrack</span>(<span class="dv">1</span>, <span class="dv">1</span>, <span class="at">r0=</span><span class="dv">0</span>, <span class="at">r1=</span>track_margin)</span>
<span id="cb17-47"><a href="#cb17-47" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb17-48"><a href="#cb17-48" aria-hidden="true" tabindex="-1"></a><span class="co"># Autotrack for the current track</span></span>
<span id="cb17-49"><a href="#cb17-49" aria-hidden="true" tabindex="-1"></a>at <span class="ot"><-</span> <span class="fu">autotrack</span>(track_position, <span class="fu">length</span>(track_list), <span class="at">r0=</span>genes_track_at<span class="sc">$</span>r1 <span class="sc">+</span> track_margin, <span class="at">margin =</span> <span class="fl">0.2</span>)</span>
<span id="cb17-50"><a href="#cb17-50" aria-hidden="true" tabindex="-1"></a><span class="co"># Plotting genes</span></span>
<span id="cb17-51"><a href="#cb17-51" aria-hidden="true" tabindex="-1"></a>kp <span class="ot"><-</span> <span class="fu">kpPlotGenes</span>(kp, <span class="at">data=</span>chchd2_data, <span class="at">r0=</span>genes_track_at<span class="sc">$</span>r0, <span class="at">r1=</span>genes_track_at<span class="sc">$</span>r1, <span class="at">gene.name.cex=</span><span class="dv">2</span>)</span>
<span id="cb17-52"><a href="#cb17-52" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb17-53"><a href="#cb17-53" aria-hidden="true" tabindex="-1"></a><span class="co"># Plotting the current track</span></span>
<span id="cb17-54"><a href="#cb17-54" aria-hidden="true" tabindex="-1"></a>kp <span class="ot"><-</span> <span class="fu">kpPlotBigWig</span>(kp, <span class="at">data=</span>bigwig.file, <span class="at">ymax=</span><span class="st">"visible.region"</span>, </span>
<span id="cb17-55"><a href="#cb17-55" aria-hidden="true" tabindex="-1"></a> <span class="at">r0=</span>at<span class="sc">$</span>r0, <span class="at">r1=</span> <span class="fl">0.20466</span>, <span class="at">col =</span> track_colour)</span>
<span id="cb17-56"><a href="#cb17-56" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb17-57"><a href="#cb17-57" aria-hidden="true" tabindex="-1"></a><span class="co"># Adjusting the y-axis limits and adding labels</span></span>
<span id="cb17-58"><a href="#cb17-58" aria-hidden="true" tabindex="-1"></a>computed.ymax <span class="ot"><-</span> <span class="dv">31</span></span>
<span id="cb17-59"><a href="#cb17-59" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb17-60"><a href="#cb17-60" aria-hidden="true" tabindex="-1"></a><span class="fu">kpAxis</span>(kp, <span class="at">ymin=</span><span class="dv">0</span>, <span class="at">ymax=</span>computed.ymax, <span class="at">numticks =</span> <span class="dv">2</span>, <span class="at">r0=</span>at<span class="sc">$</span>r0, <span class="at">r1=</span>at<span class="sc">$</span>r1, <span class="at">cex =</span> <span class="fl">1.5</span>)</span>
<span id="cb17-61"><a href="#cb17-61" aria-hidden="true" tabindex="-1"></a><span class="fu">kpAddLabels</span>(kp, <span class="at">labels =</span> <span class="fu">names</span>(track_list)[<span class="dv">1</span>], <span class="at">r0=</span>at<span class="sc">$</span>r0, <span class="at">r1=</span>at<span class="sc">$</span>r1, </span>
<span id="cb17-62"><a href="#cb17-62" aria-hidden="true" tabindex="-1"></a> <span class="at">cex=</span><span class="dv">2</span>, <span class="at">label.margin =</span> <span class="fl">0.035</span>, <span class="at">col =</span> track_colour)</span>
<span id="cb17-63"><a href="#cb17-63" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb17-64"><a href="#cb17-64" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb17-65"><a href="#cb17-65" aria-hidden="true" tabindex="-1"></a><span class="co"># E4</span></span>
<span id="cb17-66"><a href="#cb17-66" aria-hidden="true" tabindex="-1"></a>bigwig.file <span class="ot"><-</span> track_list[[<span class="st">"E4"</span>]]</span>
<span id="cb17-67"><a href="#cb17-67" aria-hidden="true" tabindex="-1"></a>track_position <span class="ot">=</span> <span class="dv">2</span></span>
<span id="cb17-68"><a href="#cb17-68" aria-hidden="true" tabindex="-1"></a>track_colour <span class="ot">=</span> <span class="st">"#6FB382FF"</span></span>
<span id="cb17-69"><a href="#cb17-69" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb17-70"><a href="#cb17-70" aria-hidden="true" tabindex="-1"></a><span class="co"># Adjusting the margin for tracks</span></span>