GSM7763806: Murine Skin CD45+ Run 2; Mus musculus; ATAC-seq
Sorted cells were pelleted by centrifugation (300xg, 5min 4°C). Supernatant was removed and cells were resuspended in 50 µl - 100 µl 0.04%BSA-PBS buffer. Cells were centrifuged (300xg, 5 min 4°C), supernatant was completely removed and chilled nuclei lysis buffer (10 mM TRIS-HCL pH7.4, 10mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 0.1% NP-40, 0.01% Digitonin, 1% BSA in nuclease-free water) was added. Lysis was performed for 2 min at 4°C, followed by addition of 50 µl washing buffer (10 mM TRIS-HCl ph7.4, 10 mM NaCl, 3 mM MgCL2, 1%BSA, 0.1% Tween-20 in nuclease-free water). After centrifugation (300xg, 5min 4°C), supernatant was removed and 45 µl chilled diluted nuclei buffer (10X Genomics) was added. After centrifugation (300xg, 5 min 4°C), supernatant was removed and 7µl chilled diluted nuclei buffer (10X Genomics) was added. Nuclei were counted (where applicable) and transposition mix was prepared with 5 µl of nuclei suspension based on the manufacturer's protocol. Transposition was performed for one hour at 37°C, followed by supplementation of master mix and beads (Single Cell ATAC Gel Beads V1.0 or V1.1 and reagents, 10X Genomics #1000175), loading on 10X Chromium Next GEM Chip H (10X Genomics #1000161) and processing on 10X Chromium Controller (10X Genomics #120212). GEM incubation was performed with 11 cycles (mouse CD45) 10-11 cycles (human CD45) of PCR. All samples were processed in duplicate runs to increase total output of cells for analysis. Library was prepared according to the protocol with cycle numbers dependent on input nuclei concentration. scATAC libraries were sequenced on an Illumina NextSeqTM 550 with NextSeqTM 500/550 High Output Kit v2.5 (75 cycles, Illumina #20024906).