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. 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.

近年来的技术进步使得大规模并行单细胞转座酶可及性染色质测序分析（single-cell Assay for Transposase Accessible Chromatin by sequencing，缩写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开展分析，但此前尚未与蛋白质水平检测相结合。
本文介绍了基于测序的抗原选择性分析联合ATAC测序技术（ATAC with Select Antigen Profiling by sequencing，缩写ASAP-seq），这是一种可同时对数千个单个细胞的可及染色质与蛋白质水平进行表征的工具：它将稀疏的scATAC数据与数百种细胞表面及胞内蛋白标志物的高灵敏可靠检测相结合，还可选择性富集线粒体DNA覆盖度以用于谱系示踪（mtscATAC-seq），且对ATAC-seq数据质量的影响极小。
ASAP-seq采用新型桥接策略，可利用已针对CITE-seq及相关技术开发的商用抗体-寡核苷酸偶联物。我们在造血分化、外周血单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分析场景中验证了ASAP-seq的实用性。
本研究从3名生物学供体中分选人T细胞，对其进行激活，并利用CRISPR/Cas9系统对靶基因进行扰动。通过靶向CD4（HTO2）、ZAP70（HTO4）、NFKB2（HTO5）、CD3E（HTO3+HTO13）、CD3E+CD4（HTO3+HTO12）的向导RNA（gRNA）以混合池化方式构建基因敲除模型，同时设置2个非靶向对照（NTCs；HTO1）。针对CD4、ZAP70、NFKB2及非靶向对照组别，我们使用HTO12与HTO13对不同的gRNA重复样本进行编码，最终实现了单个细胞中染色质可及性与蛋白质丰度变化的同步检测。