GSM9364535: SUDHL5 cells, IgG CUT&RUN, bio rep 2; Homo sapiens; OTHER
Briefly, cells were collected and washed by PBS. After final wash, nuclei were isolated using nuclei extraction buffer (20mM HEPES pH 7.9, 10 mM KCl, 0.1% Triton X-100, 20% glycerol, 1x cOmplete protease inhibitors (Roche 11836153001), 0.5mM spermidine) and then washed twice with wash buffer (20mM HEPES pH 7.5, 150mM NaCl, 0.5mM Spermidine, 1x cOmplete protease inhibitor). Five-hundred thousand nuclei were counted and incubated with activated Concanavalin A coated beads (EpiCypher 21-1411) and subsequently mixed with BCL6 antibodies (Cell Signaling D65C10) in antibody buffer (wash buffer + 0.01% digitonin + 2 mM EDTA) for overnight incubation on nutator in a cold room. IgG controls (normal rabbit IgG, Cell Signaling 2729) were set in parallel for antibody enrichment and specificity validation. Nuclei were then washed three times with digitonin buffer (wash buffer + 0.01% digitonin) and incubated with pAG-MNase (EpiCypher 15-1116) for 10 minutes at room temperature. After 10 minutes of incubation, nuclei were washed three times again with digitonin buffer and resuspended with prechilled low-salt, high-Ca2+ buffer (20 mM HEPES pH 7.5, 0.5mM spermidine, 1x cOmplete protease inhibitor, 10mM CaCl2) to initiate 1-hour digestion at 0°C using an ice block. The reaction was terminated by switching into stop buffer (340 mM NaCl, 20mM EDTA, 4 mM EGTA, 50 µg/mL RNaseA, 50 µg/mL glycogen) and incubating at 37°C for 10 minutes to release digested DNA from the nuclei. DNA was then purified with Ampure beads (Beckman A63882), subjected to two-sided size selection(0.5X to remove large fragment and 2X to recover desired nucleosomal DNA fragments). Sequencing libraries were generated with KAPA Hyper Prep Kits (Roche KK8502) using 14 cycles of PCR amplification. Libraires were validated on a Tapestation 4200 (Agilent G2991BA), quantified by Qubit High Sensitivity dsDNA Kit (Life Technologies Q32854)