High throughput joint profiling of chromatin accessibility and protein levels in single cells [Tcell KO]. High throughput joint profiling of chromatin accessibility and protein levels in single cells [Tcell KO]

Recent technological advances have enabled massively parallel single-cell Assay for Transposase Accessible Chromatin by sequencing (scATAC-seq) to simultaneously profile the epigenomic landscape in thousands of individual cells. scATAC-seq methods sample genomic DNA accessible to transposases, but have not previously been combined with measurement of protein levels. Here, we present ATAC with Select Antigen Profiling by sequencing, ASAP-seq, a tool to simultaneously profile accessible chromatin and protein levels in thousands of single cells, pairing sparse scATAC data with robust detection of hundreds of cell surface and intracellular protein markers, and optionally, enriched mtDNA coverage for lineage tracing (mtscATAC-seq) with minimal impact on ATAC-seq data quality. ASAP-seq makes use of a novel bridging approach to utilize existing commercially available antibody:oligo conjugates developed for CITE-seq and related technologies. We demonstrate the utility of ASAP-seq in the context of hematopoietic differentiation, cell surface marker dynamics following peripheral blood mononuclear cell stimulation, and as a combinatorial decoder of multiplexed perturbations in primary T cells. Overall design: Sorted human T-cells from 3 biological donors were activated and target genes were perturbed using CRISPR / Cas9. Gene KOs were generated in a pooled fashion using gRNAs targeting CD4 (HTO2), ZAP70 (HTO4), NFKB2 (HTO5), CD3E (HTO3 + HTO13), CD3E+CD4 (HTO3 + HTO12) and 2 non-targeting controls (NTCs; HTO1). Different gRNA replicates were encoded using HTO12 and HTO13 for CD4, ZAP70, NFKB2, and NTC conditions. Changes in chromatin accessibility and protein abundance were determined concomitantly in single cells.

近年来的技术进步已使得大规模并行单细胞转座酶染色质可及性测序（single-cell Assay for Transposase Accessible Chromatin by sequencing, 简称scATAC-seq）能够同时对数千个单个细胞的表观基因组图谱进行表征。scATAC-seq技术通过捕获转座酶可及的基因组DNA进行测序，但此前尚未与蛋白质水平检测实现联合应用。本研究介绍了结合选择性抗原谱分析的测序技术（ATAC with Select Antigen Profiling by sequencing, 简称ASAP-seq），该工具可同时对数千个单个细胞的染色质可及性与蛋白质水平进行分析，能够将稀疏的scATAC测序数据与数百种细胞表面及细胞内蛋白质标志物的稳健检测相结合，还可选择性引入用于谱系示踪的富集线粒体DNA覆盖度分析（mtscATAC-seq），且对ATAC-seq数据质量的影响极小。ASAP-seq采用了一种新型桥接策略，可利用现有为CITE-seq及相关技术开发的商业化抗体-寡核苷酸偶联物。我们分别在造血分化、外周血单个核细胞刺激后的细胞表面标志物动态变化，以及原代T细胞多重扰动的组合解码这三个场景中验证了ASAP-seq的实用性。实验设计概述：从3名生物学供体中分选得到的人T细胞经激活后，通过CRISPR/Cas9系统对靶基因进行扰动。本研究通过靶向CD4（HTO2）、ZAP70（HTO4）、NFKB2（HTO5）、CD3E（HTO3 + HTO13）、CD3E+CD4（HTO3 + HTO12）的向导RNA（gRNA）以及2种非靶向对照（NTCs；HTO1）以混合池方式构建基因敲除（KO）模型。针对CD4、ZAP70、NFKB2及NTC组，使用HTO12和HTO13对不同gRNA重复样本进行编码。最终可在单个细胞中同时检测染色质可及性与蛋白质丰度的变化。