GSM8499428: Mutu1, H3K27ac, rep1; Homo sapiens; OTHER
500,000 cells per condition were crosslinked using 0.1% formaldehyde for 1 minute, then crosslinking was quenched using 0.125M glycine. Cells were spun down 600g 3 minutes, washed twice with XL Wash Buffer at RT. Activated ConA beads were added to the cells and incubated on nutator for 10 minutes. Beads were then collected on a magnet and 50uL/reaction of fresh ice-cold Antibody Buffer was added to the beads. 1ug of each antibody was added to the reaction and incubated o/n on a nutator at 4°C with cap elevated. Next day beads were collected on a magnet, washed twice with 200uL of ice-cold Digitonin Buffer, resuspended in 50 uL of the same buffer and 2.5 uL of pAG-MNase was added to each reaction. Samples were incubated for 10 minutes at RT on nutator with caps elevated. Beads were washed again twice with ice-cold Digitonin Buffer and resuspended in 50 uL of the same buffer. 1 uL of 100 mM CaCl2 was added to each sample on ice and samples were incubated for 2 hours on nutator at 4°C with caps elevated. Chromatin digestion with MNase was stopped using Stop Buffer at 37°C for 10 minutes in a thermocycler. Supernatant was incubated with 0.8 uL of SDS and 1 uL of Proteinase K o/n at 55°C in a thermocycler to revert crosslinking. DNA was then purified using 1.1X AMPure XP Beads and quantified using Qubit 1X dsDNA HS quantification kit Libraries were prepared using NEBNext Ultra II DNA Library Prep Kit