GSM6339326: sciPlexATAC3_HumanPBMC_MLR_DrugScreen; Homo sapiens; ATAC-seq
For all experiments, cells were harvested following the sciPlex-ATAC-seq protocol as described in the methods section of our manuscript. Nuclei were isolated in-well and barcoded by fixation of well-specific barcode containing oligonucleotides. After fixation wells were pooled and nuclei pellets flash frozen until sciPlex-ATAC-seq library preparation. Nucle were subjected to two- or three-level sciPlex-ATAC-seq following the protocols described in detail in the methods section of our manuscript. Briefly: Two-level library construction: To capture poly-adenylated hash molecules within the context of sciATAC-seq, we modified the original sciATAC-seq protocol (Cusanovich et al., Science, 2015), to include an indexed primer extension step. The indexed extension of the hash molecules is followed by indexed transposition within the reaction same well, creating a known pairing between well barcodes of hash and tagmented genomic DNA (gDNA). By design, extended hash molecules and tagmented gDNA possess nextera P5 and P7 PCR handles. After tagmentaiton, nuclei are pooled, stained with DAPI, and flow-sorted into 96 well plates for crosslink reversal and PCR. Amplification with indexed PCR primers adds an additional level of barcoding to both hash labels and accessible chromatin fragments. This protocol leverages unique combinations of well barcodes on hash and chromatin fragments to pair single cell chromatin profiles with their corresponding hash identifiers. Three-level library construction:  Accessible chromatin within all nuclei is tagmented using a single commercially-available transposome complex containing universal Nextera adaptors. Two separate splinted ligation reactions are then used to sequentially add well-specific barcodes separately to each side of the transposed DNA fragments, followed by a final PCR-based barcode addition (Domcke et. al. Science, 2020). In these three-level experiments, hash oligos bearing the universal Nextera P7 sequence are annealed to a capture oligo possessing the Nextera P5 sequence prior to ligation steps, and thus resemble transposed genomic DNA. Therefore, annealed nuclear labels and chromatin fragments can undergo the exact same indexing reactions as chromatin within each nucleus.