GSM5608672: ChIP-Seq_HeLa_YY1_siCTL; Homo sapiens; ChIP-Seq
For ChIP assays, cells were then fixed with 1% formaldehyde (Sigma) for 10 mins at room temperature (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. Resultant was immunoprecipitated with anti-PHF8 or anti-YY1 antibody overnight at 4 ℃, followed by incubation with protein G magnetic beads for an additional 2 hrs. After washing and elution, the protein-DNA complex was reversed by heating at 65 ℃ overnight. Immunoprecipitated DNA was purified by using QIAquick spin columns (Qiagen) and subjected to high throughput sequencing. The libraries were constructed following NEB'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.