GSM5515081: HiChIP WT3; Homo sapiens; OTHER
Cells were detached from tissue culture plates with Acctuase. 25x106 cells were crosslinked, lysed, and digested with MboI (NEB) for 2 hours at 37°C. Each sample was split into two reactions to ensure efficient contact generation. Biotin was incorporated onto DNA ends for 1 hour at 37°C, and then ligated with T4 ligase overnight at room temperature. Nuclei were pelleted, resuspended, and sonicated using a Covaris E220. At this point samples were combined back together. Proximity ligations bound to the cohesion complex were enriched by incubating overnight at 4°C using an SMC1A antibody (Bethyl). Biotinylated DNA was pulled out with Streptavidin C-1 beads (Thermo Fisher), which were then subjected to transposition with 1μL Tn5 (Illumina). DNA was amplified with PCR for 5 cycles and size selected with Ampure XP beads (Beckman Coulter). Cohesin HiChIP