GSM2975777: U2OS_control; Homo sapiens; ChIP-Seq
2x10^6 cells were inoculated on 10 cm2 dish 2 days before experiments. At the appointed experimental points a two-step cross-linking procedure was applied for an efficient fixation of NF-κB dimers with DNA [27]. Cells were washed twice with PBS, a solution of DSG (disuccinimidyl glutarate) in PBS was added to the final concentration of 2 mM, and then cells were incubated for 45 minutes at RT with rotation. Next, cells were washed twice with PBS and the second fixation was performed using 1% formaldehyde for 10 min at RT. 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 15 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 3µl/sample of anti-RelA(p65) polyclonal antibody (C15310256, Diagenode) or without antibody (mock-IP), according to the manufacturer protocol. Immunoprecipitated DNA fragments and input DNA were sequenced using the HiSeq 1500 system with TruSeq workflow (Illumina)
GEO
GSM2975777