GSM9745144: Mixed, barcode-transduced primary bulk human T cells, in vitro, donor 2, scATAC library; Homo sapiens; ATAC-seq
After sorting, cells were pooled, washed once in cold PBS and then lysed in Lysis Buffer (Nuclease-Free H2O with 10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.1% NP40, 0.1% Tween20, and 0.01% Digitonin) for the following times: 5 mins (cell lines), 3 mins (in vitro primary cells), 3 mins 45 seconds (ex vivo primary cells). Cells were then resuspended in 1X Nuclei Buffer and analyzed for nuclei count and lysis efficiency via 0.4% Trypan Blue staining on the Countess 3 (ThermoFisher), with >95% lysis efficiency typical. Cells were then diluted to ≤2.4x106 cells/mL for capture of ~8000 nuclei in a single 10X capture well, following manufacturer protocols for Chromium Next GEM Single Cell ATAC Reagent Kits v2 (10X Genomics, User Guide CG000496). Two modifications were required to this protocol for barcode enrichment, as described previously19: first, 1.32µL of 50µM spike-in barcode enrichment oligo was added to the GEM reaction master mix (step 2.1a in 10X protocol); second, the PCR following GEM generation was increased from 12 to 15 cycles (step 2.5a in 10X protocol). Libraries were prepared per manufacturer specifications and analyzed using the TapeStation (Agilent) and Qubit Fluorometer (ThermoFisher). Final scATAC and barcode libraries were pooled to 10nM concentration for sequencing. Final concentrations for individual libraries in the pool were calculated to achieve a target sequencing depth of 40,000 reads/nuclei (scATAC library) and 1,000 reads/nuclei (barcode library).