GSM4838758: MCF7 cells, KO#2 (clone with HSF1 knockout)_estrogen_1h; Homo sapiens; ChIP-Seq
At the appointed experimental points culture medium was removed and fixation was performed using 1% formaldehyde in PBS for 12 min at RT with rotating. Formaldehyde fixation was quenched by glycine (125 mM final concentration) and then nuclei were isolated using buffers and protocol from iDeal ChIP-seq Kit for Transcription Factors (Diagenode). Chromatin of nuclei re-suspended in 200 µl was sheared using Bioruptor® PLUS combined with the Bioruptor® Water cooler & Single Cycle Valve (at HIGH power setting) with 20 cycles of 30 sec shearing followed by 30 sec of standby; chromatin fragments with approximate length 100-600 bp were obtained. Chromatin immunoprecipitation was carried out using the iDeal ChIP-seq Kit for Transcription Factors (Diagenode) with 30μg of chromatin and 4µl/sample of anti-ERalpha mouse monoclonal antibody (Diagenode, C15100066) or without antibody (mock-IP), according to the manufacturer protocol. Immunoprecipitated DNA fragments from four biological ChIP replicates was pooled. ChIP samples and input DNA (mixed input DNA of all modified cell lines and treatments) were sequenced using the HiSeq 1500 system with TruSeq workflow (Illumina)