GSM6927654: CUTandRUN_220310_K562-CAS-IgG; Homo sapiens; OTHER
Cells were resuspended in Wash Buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 1 x Roche complete EDTA-free protease inhibitor). Cells were bound to activated Concanavalin A beads. Bead-bound cells were resuspended in Anitbody Buffer (Wash Buffer supplemented with 2 mM EDTA and 0.05% Digitonin) and incubated with the antibody for 2h at room temperature. Samples were washed in Digitonin Buffer (Wash Buffer supplemented with 0.05% Digitonin) then incubated in Digitonin Buffer with pAG-MNase enzyme (cell signaling) for 1h at room temperature. Samples were washed. Then MNase was activated by the addition of CaCl2 (50 μL of Digitonin Buffer supplemented with 4mM CaCl2). Samples incubated at 1h at 37ºC. DNA digestion was stopped by STOP Buffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 0.1% Digitonin, 100 μg/mL RNAse A, 50 μg/mL Glycogen) and incubated at 37ºC for 1h. DNA was purified with MinElute PCR purification Kit (Qiagen). Sequencing libraries were prepared from eluted DNA using Rubicon ThruPLEX DNA-seq kit.