GSM6430170: Multiome - ATAC - Patient 21; Homo sapiens; ATAC-seq
Frozen PBMCs were thawed in a 37°C water bath for 3-5 min until no ice was visible. Cells were washed twice with 1 mL PBS + 0.04% BSA and pelleted (300×g for 5 min at 4°C). Dead cells were removed according to the Demonstrated Protocol: Removal of Dead Cells from Single Cell Suspensions for Single Cell RNA Sequencing (10X Genomics). Using the MACS Dead Cell Removal Kit (Miltenyi Biotec), the pellet was resuspended in 100 mL Dead Cell Removal MicroBeads and incubated for 15 minutes at room temperature. After incubation, the cell suspension was diluted with 1X Binding Buffer and applied to a MS column. The dead cells were retained on the column and the live cells passed through the column and were collected. After dead cell removal, the samples were washed twice with 1 mL PBS + 0.04% BSA and the cell concentration was determined using a Cellometer K2 cell counter (Nexcelom Biosciences). Nuclei were isolated according to the Demonstrated Protocol: Nuclei Isolation for Single Cell Multiome ATAC + Gene Expression (10x Genomics, CG000365 Rev A). Briefly, cells were added to a 2.0 mL low binding tube and centrifuged (300×g for 5 min at 4°C) using a swinging bucket rotor. The supernatant was removed, and the cell pellet was resuspended in 100 µL of chilled 10x Genomics Lysis Buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 0.1 % NP-40 Substitute, 0.01% digitonin, 1% BSA, 1 mM DTT, 1 U/mL RNase inhibitor 40 U/mL) by pipette-mixing 10 times. Cells were incubated on ice for 3 min, followed by dilution with 1 mL of chilled Wash Buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 1% BSA, 1 mM DTT, 1 U/mL RNase inhibitor 40 U/mL). Nuclei were then centrifuged (500×g for 3 min at 4°C), and the supernatant was slowly removed. The nuclei were washed one additional time with 1 mL Wash Buffer. Nuclei were resuspended in chilled Nuclei Buffer (1X Nuclei Buffer (10x Genomics), 1 mM DTT, 1 U/mL RNase inhibitor 40 U/mL); the concentration was determined using a Cellometer K2 cell counter (Nexcelom Biosciences) and the samples were adjusted to a concentration appropriate for our targeted nuclei recovery. The single-cell ATAC library construction and gene expression library construction were carried out as described in the Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide (CG000338 Rev A). ATAC and GEX libraries were sequenced separately on an HiSeq 4000 (Illumina) before demultiplexing, alignment to the reference genome, and post-alignment quality control.