GSM5034245: HCT116-NUP93-mAC,Cut&Run for BRD4,UT, biological replicate 1; Homo sapiens; OTHER
0.5 million live HCT116 or HeLa cells were harvested and resuspended in 100ul wash buffer [20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, supplemented with Protease Inhibitor EDTA-Free tablet (Roche 11836170001)]. Activated Concanavalin A (EpiCypher 21-1401) was incubated with cells at room temperature for 10min to let the cells bind to the beads. For each target protein factor, 0.5ug antibody was added to each sample and incubated in the antibody buffer (wash buffer + 0.01% Digitonin and 2 mM EDTA) at 4ºC for 4-6hr. The beads were then washed twice with digitonin buffer [wash buffer + 0.01% Digitonin], and 2.5 µL pAG-MNase (EpiCypher, 15-1116) was added to each sample. After ten minutes of incubation at room temperature, excessive pAG-MNase was washed out by a two-time digitonin buffer wash. Then targeted chromatin was digested and released from cells by 2 hours of incubation with the presence of 2 mM CaCl2 at 4ºC, which were collected from the supernatant, were subjected to phenol/chloroform DNA extraction, sequencing library construction using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina (NEB, E7645L).
GEO Accession
GSM5034245