GSM6793419: AS10-21035, Control, scATACseq; Homo sapiens; ATAC-seq
Thawed PBMCs were washed with PBS/0.04% BSA. Cells were counted and 100,000-1,000,000 cells were added to a 2 mL-microcentrifuge tube. Cells were centrifuged at 300xg for 5min at 4°C. The supernatant carefully completely removed, and 0.1X lysis buffer (1x: 10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, nuclease-free H2O, 0.1% v/v NP-40, 0.1% v/v Tween-20, 0.01% v/v digitonin) was added. After 3 min incubation on ice, 1 ml of chilled wash buffer was added. The nuclei were pelted at 500 xg for 5 min at 4°C and resuspended in a chilled diluted nuclei buffer (10X Genomics) for scATAC-seq. Nuclei were counted and the concentration was adjusted to run the assay. ScATAC-seq was performed immediately after nuclei isolation and following the Chromium Single Cell ATAC Reagent Kits V1.1 User Guide (10x Genomics, Pleasanton, CA). Transposition was performed in 10 ul at 37°C for 60 min on at least 1,000 nuclei, before loading of the Chromium Chip H (PN-2000180). Barcoding was performed in the emulsion (12 cycles) following the Chromium protocol. After post GEM cleanup, libraries were prepared following the protocol and were indexed for multiplexing (Chromium i7 Sample Index N, Set A kit PN-3000427). Each library was assessed on a Bioanalyzer (High-Sensitivity DNA Bioanalyzer kit).