GSM8728126: Karpas422-IgG-CR; Homo sapiens; OTHER
CUT&RUN was performed with minor modifications of the CUTANA (EpiCypher) protocol. Briefly, cells were fixed with 0.1% formaldehyde in PBS for 1 minute, followed by quenching with 125 mM glycine. Cells were washed with cold Wash Buffer (20 mM HEPES-KOH, pH 7.9, 150 mM NaCl, 0.5 mM Spermidine, protease inhibitor cocktail) and then bound to concavalin A beads for 10 minutes at room temperature. Supernatant was removed and beads were resuspended in Antibody Buffer (20 mM HEPES-KOH, pH 7.9, 150 mM NaCl, 0.5 mM Spermidine, 0.01% Digitonin, 2 mM EDTA, protease inhibitor cocktail) along with primary antibody. Samples were incubated overnight in the cold room, rotating. Beads were then resuspended in cold Digitonin Buffer 20 mM HEPES-KOH, pH 7.9, 150 mM NaCl, 0.5 mM Spermidine, 0.01% Digitonin, protease inhibitor cocktail), washed twice, then incubated with CUTANA pAG-MNase (Epicypher) for 10 minutes at RT. Beads were then washed twice with cold Digitonin buffer, resuspended in Digitonin Buffer with 50 mM CaCl2, and samples incubated for two hours with rotation at 4 °C. MNase was quenched by addition of STOP Buffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 50 mg/ml RNase, 50 mg/ml Glycogen) and incubation for 10 mins at 37 °C. 0.1% SDS and Proteinase K was added to the supernatant and incubated for one hour at 55 °C followed by two hours at 65 °C. DNA was purified using Phenol-Chloroform Isoamyl Alcohol mix (Millipore) and PhaseLock tubes (5PRIME), and quantified using a Qubit 4 Fluorometer (Thermo) prior to proceeding with library preparation. ChIP and CUT&RUN libraries were prepared using NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB) according to the manufacturer's instructions. All libraries were analyzed by TapeStation prior to sequencing. Single-end sequencing was performed on the Illumina NextSeq 500 sequencer.