GSM9201757: CR028T, CR029T, CR030T, CR031T; Homo sapiens; ATAC-seq
Cryopreserved cells were thawed, and 100k cells were transferred to a microtube, and centrifuged at 1,000 g for 5 min at 4 °C. The pellet was washed twice with 200 µL of 0.04% BSA in PBS and resuspended in 50 µL of the same buffer. A 5 µL aliquot was used for cell counting on a Cellometer Auto 2000 (Nexcelom Biosicence) with AO/PI staining. Equal numbers of cells from 2-4 individual donors were pooled to prepare a total of 200k cells, filtered through a 40 µm Flowmi strainer (SP Bel-Art, 136800040), and centrifuged again. The pellet was resuspended in 90 µL of cold 0.1× Lysis Buffer, containing 10mM Tris-HCl (pH7.4), 10 mM NaCl, 3 mM MgCl2, 0.01% Tween-20, 0.01% Nonidet P40 Substitute (Sigma-Aldrich, 74385), 0.001% digitonin (Thermo Fisher Scientific, BN2006), and 1% BSA, with gentle pipetting and incubated on ice for 5 min. Following incubation, 100 µL of Wash Buffer with 10mM Tris-HCL (pH7.4), 10mM NaCl, 3mM MgCl2, 1% BSA, and 0.1% Tween-20, was added, the sample was centrifuged, and the supernatant was removed. The pellet was then washed twice with gentle pipetting and resuspended in 100 µL of 1× Nuclei Buffer (10x Genomics). After centrifugation, 80uL of supernatant was removed, leaving 20 uL to minimize loss, and 5 uL was used for cell counting. A total of 25k nuclei ware prepared for tagmentation. Libraries were prepared with 10X Genomics Chromium Single Cell ATAC Library & Gel Bead Kit v1 following manufacturer's instructions.