GSM6857439: HCT116 AsiSI WT_rH2AX; Homo sapiens; ChIP-Seq
For each precipitation, 1 × 10^7 cells were crosslinked by the addition of formaldehyde directly to the growth medium to a final concentration of 1%. After 10 min, crosslinking was quenched by the addition of glycine to a final concentration of 0.25 M at room temperature. Crosslinked cells were washed with PBS, scraped, and washed again with PBS containing 1 mM EDTA, and then lysed gently on ice for 5 min using 0.5 mL of ice-cold NP-40 lysis buffer. Cell lysates were added on top of a 1.25 mL sucrose cushion (24% sucrose (wt/vol) in NP-40 lysis buffer) and centrifuged at 12,000 rpm for 10 min at 4 °C to isolate the nuclei pellet. The chromatin pellet was washed twice with 1 mL PBS/1 mM EDTA, followed by centrifugation at 12,000 rpm for 1 min at 4 °C. The supernatant was discarded, the tube was pre-warmed at 37 °C for 2 min, and then 40 U MNase was added followed by incubation at 37 °C for 15 min with rotation at 700 rpm. Subsequently, the MNase was inactivated by the addition of 20 μL 0.5 M EDTA (to a final concentration of 10 mM) and 40 μL 0.5 M EGTA (to a final concentration of 20 mM). The mixture was then placed on ice, mixed thoroughly by pipetting, and sonicated using pre-cooled Biorupter at 15–20 x (30 s on/30 s off) on high position. The sonicated chromatin was spun down at 12,000 rpm for 10 min at 4 °C to collect the supernatant chromatin. Sequencing libraries were prepared using 10 ng of purified DNA (average size 200–400 bp) using the NEBNext Ultra II Library Prep Kit for Illumina (E7645S, New England Biolabs)