Single-nucleus chromatin accessibility profiling identifies cell types and functional variants contributing to major depression

We performed high-throughput snATAC-seq using the 10X Genomics Chromium platform on archived post-mortem dorsolateral prefrontal cortex (BA9) tissue in MDD subjects who died by suicide and neurotypical control subjects to identify cell-type specific differentially accessible chromatin regions. We further combined snATAC-seq with previously generated snRNA-seq data from the same subjects to generate high-resolution multi-modal accessibility and expression atlas of cortical cells. This identified accessible chromatin regions potentially regulating expression of genes. Further, MDD-associated genetic risk variants were examined for their allele-specific effects on chromatin accessibility and transcription factors binding sites in cell type speciifc manner, elucdiating target risk genes and pathways associated with MDD. 10X Genomics Chromium snATAC-seq was performed on nuclei extracted from BA9 of the post-mortem brain tissue of 44 MDD cases who died by suicide and 40 neurotypical controls. We multiplexed nuclei obtained from a pair of male (n=42) and female (n=42) subjects before 10x microfluidic capture and produced combined libraries. These libraries were demultiplexed using common variants and sex-specific chromatin accessibility signals. Unsupervised clustering was perfomed using accessibility measured in 500bp bins and pseudobulk differential accessibility analysis was performed between cases and controls for each cluster and each broad brain cell type. The library preparation was performed with the Chromium Single Cell ATAC v1.1 Chemistry. Paired-end sequencing was performed on the Illumina NovaSeq 6000. FASTQ files were produced with 10x cellranger-atac mkfastq. Alignments were generated with refdata-cellranger-arc-GRCh38-2020-A-2.0.0 and cellranger-atac count. 10x fragment files were converted to peak count matrices using ArchR [release_1.0.1].

本研究借助10X Genomics Chromium平台，对重度抑郁症（Major Depressive Disorder, MDD）自杀死亡受试者及神经典型对照受试者的存档死后背外侧前额叶皮层（BA9）组织开展高通量单细胞核转座酶可及性测序（single-nucleus Assay for Transposase-Accessible Chromatin using sequencing, snATAC-seq），以鉴定细胞类型特异性差异可及染色质区域。
我们进一步将snATAC-seq数据与同一受试者的既往单细胞核RNA测序（single-nucleus RNA sequencing, snRNA-seq）数据相结合，构建了高分辨率的皮层细胞多模态可及性与表达图谱。该图谱鉴定出潜在调控基因表达的可及染色质区域。此外，我们针对MDD相关遗传风险变异，以细胞类型特异性方式分析其对等位基因特异性染色质可及性及转录因子结合位点的影响，阐明了与MDD相关的潜在风险靶点基因及通路。
本研究对44名自杀死亡的MDD病例及40名神经典型对照受试者的死后大脑BA9组织提取的细胞核进行10X Genomics Chromium snATAC-seq实验。在10x微流体捕获前，我们将来自42名男性及42名女性受试者的细胞核进行多重标记并构建混合文库。随后通过常见变异及性别特异性染色质可及性信号对混合文库进行解多重处理。
研究以500bp窗口内的可及性信号开展无监督聚类，并针对每个聚类及每类宽泛脑细胞类型，在病例组与对照组之间进行伪bulk差异可及性分析。
文库制备采用Chromium单细胞ATAC v1.1试剂盒。测序在Illumina NovaSeq 6000平台上以双端模式完成。测序数据通过10x Cell Ranger ATAC mkfastq生成FASTQ文件。比对分析借助refdata-cellranger-arc-GRCh38-2020-A-2.0.0参考数据集及Cell Ranger ATAC count流程完成。最后将10x片段文件转换为峰计数矩阵，所用工具为ArchR软件（版本release_1.0.1）。