High Capacity Sample Multiplexing for Chromatin Accessibility Profiling Within Single Cells

Single-cell chromatin accessibility has emerged as a powerful means of understanding the epigenetic landscape of diverse tissues and cell types, but profiling cells from many independent specimens is challenging and costly. Here we describe a novel approach, sciPlex-ATAC-seq, which uses unmodified DNA oligos as sample-specific nuclear labels, enabling the concurrent profiling of chromatin accessibility within single nuclei from virtually unlimited specimens or experimental conditions. We first demonstrate our method with a chemical epigenomics screen, in which we identify drug-altered distal regulatory sites predictive of compound- and dose-dependent effects on transcription. We then analyze cell type-specific chromatin changes in PBMCs from multiple donors responding to synthetic and allogeneic immune stimulation. We quantify stimulation-altered immune cell compositions and isolate the unique effects of allogeneic stimulation on chromatin accessibility specific to T-lymphocytes. Finally, we observe how various immunosuppressive compounds disrupt canonical allogeneic activation of resting T-cells through chromatin. Overall design: Single cell ATAC-seq libraries were produced using two and three level single-cell combinatorial indexing ATAC sequencing (sci-ATAC-seq) from small molecule-inhibited or untreated A549, and NIH-3T3 cells, as well as from cultured human PBMCs. PBMCs were cultured in the presense of irradiated foreign PBMCs (mixed lymphocyte reactions), T-cell expansion beads, or otherwise untreated. Irradiated PBMCs were also profiled. For individual experiments, different cells and treatments were hashed and pooled prior to sci-ATAC-seq using a nuclear barcoding strategy. This nuclear barcoding strategy relies on fixation of barcode containing, well-specific oligos that are specific to a given cell type, replicate or treatment condition.