GSM6589206: HCT116, ASXL2 KO, MLL4; Homo sapiens; ChIP-Seq
Cells were cross-linked with 1% formaldehyde for 10 min. Crosslinking reaction was stopped by 125 mM glycine for 10 min. 30 – 40 million cross-linked cells were resuspended in 5 mL lysis buffer (5 mM PIPES, pH 7.5, 85 mM KCl, 1% NP-40 and protease inhibitors), incubated on ice for 10 min, and spun down at 1,000 g for 5 min at 4°C. The pellet was washed once with 5 mL lysis buffer. Resulting nuclei were resuspended with 2 mL TE buffer (50 mM Tris-HCl, pH8.0, 10 mM EDTA and protease inhibitors) and sonicated for 10 min (30 sec on and 30 sec off) at 20% amplitude. After adding detergents to a final concentration of 1% Triton-X100, 0.1% SDS, 0.1% sodium deoxycholate (1x RIPA), sonicated chromatin was spun down at 15,000 g for 10 min at 4°C. For each ChIP, 10 µg of target antibodies and 2 μg of spike-in antibody (anti-H2Av, Active Motif, #61686) were mixed with 400 – 600 mg sonicated chromatin and incubated on a rotator at 4°C overnight. Next day, 50 μL prewashed protein A Dynabeads (Thermo Fisher) were added to chromatin-antibody complex and incubated for 2 hours at 4°C. ChIPed samples were washed twice with 1 mL RIPA, twice with 1 mL RIPA + 300mM NaCl, twice with 1 mL LiCl buffer and once with PBS. Samples were eluted in 100 μL buffer containing 0.1M NaHCO3, 1% SDS, and 20μg Proteinase K at 65°C overnight and DNA was purified using QIAquick PCR purification kit (Qiagen). Libraries were prepared according to NEB (Illumina kit) instructions. DNA were used to construct libraries using NEBNext® Ultra™ II DNA Library Prep kit with AMPure XP magnetic beads (Beckman Coulter).