GSM5658232: BRD4-E2-MCF7; Homo sapiens; ChIP-Seq
Cells were fixed with disuccinimidyl glutarate (DSG) (2 mM) (Proteochem) for 45 mins at RT, washed twice with PBS and then double-fixed with 1% formaldehyde for another 10 mins at RT. Fixation was stopped by adding glycine (0.125 M) and incubated for 5 mins at RT, followed by washing with PBS twice. Chromatin DNA was sheared to 300~500 bp average in size through sonication. The resultant protein was immunoprecipitated with anti-BRD4 antibody overnight at 4 °C, followed by incubation with protein G magnetic beads (Bio-Rad) for an additional 4 hrs. After washing and elution, the protein-DNA complex was reversed by heating at 65 °C overnight. Immunoprecipitated DNA was purified by using QIAquick spin columns and subjected to high throughput sequencing. The libraries were constructed following Illumina's Chip-Seq Sample prep kit. Briefly, Chip DNA was end-blunted and added with an 'A' base so the adaptors from Illumina with a 'T' can ligate on the ends. Then 200–400 bp fragments are gel-isolated and purified. The library was amplified by 18 cycles of PCR.