GSM8219602: SK-L008-GM12868-ATAC_Kethoxal_biotin_pulldown-2; Homo sapiens; OTHER
Cells resuspended in 500 uL of media were supplemented with 5 mM N3-kethoxal (final concentration), then incubated for 10 minutes at 37C with shaking at 500 rpm in a Thermomixer, then pelleted by centrifuging at 500 g for 5 minutes at 4C. The ATAC-seq step was then carried out, by resuspending cells in 50 uL ATAC-RSB-Lysis buffer, incubation on ice for 3 minutes, addition of 1 mL ATAC-RSB-Wash buffer, centrifugation at 500 g for 5 min at 4C, and transposition in a total volume of 50 uL. DNA purification was carried out using the MinElute PCR Purification Kit. Multiple parallel ATAC reactions were carried out, including ~50,000 cells each, then were pooled together for the click reaction and biotin pull down. Click reaction was carried out by combining transposed DNA, 5 uL 20 mM DBCO-PEG4-biotin (DMSO solution, Sigma 760749), 20 mM K3BO3 (pH 7.0) and 20 uL 10XPBS in a final volume of 200 uL. Biotinylated DNA was purified using the MinElute PCR Purification Kit. For streptavidin pulldown of biotin-labeled DNA, 10 uL of 10 mg/mL Dynabeads MyOne Streptavidin T1 beads (Life Technologies, 65602) were separated on a magnetic stand, then washed with 300 uL of 1X TWB (Tween Washing Buffer; 5 mM Tris-HCl pH 7.5; 0.5 mM EDTA; 1 M NaCl; 0.05% Tween 20). The beads were resuspended in 300 uL of 2X Binding Buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA; 2 M NaCl), the biotinylated DNA was added (diluted to a final volume of 300 uL if necessary), and the beads were incubated for >15 minutes at room temperature on a rotator. After separation on a magnetic stand, the beads were washed with 300 uL of 1X TWB, and heated at 55C in a Thermomixer with shaking for 2 minutes. After removal of the supernatant on a magnetic stand, the TWB wash and 55C incubation were repeated. Libraries were prepared on beads using the same settings as for ATAC-seq (72C for 3 minutes, 98C for 30 seconds, 10 cycles of 98C for 10 seconds, 63C for 30 seconds, 72C for 30 seconds). Beads were separated on a magnetic stand, and the supernatant was cleaned up using the MinElute PCR Purification Kit.