GSM4314797: H9_P4CNCC_CTCF_ChIPseq_replicate1; Homo sapiens; ChIP-Seq
5-15 million cells were cross-linked per ChIP experiment in 2mL PBS per 6-well with 1% methanol-free formaldehyde for 5-10 min and quenched with a final concentration of 0.125M glycine for 5 min with nutation. Cross-linked cells were washed with PBS, scraped and pelleted by centrifugation, flash-frozen in liquid nitrogen and stored at -80°C. Samples were defrosted on ice and resuspended in 5mL LB1 (50 mM Hepes-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, with 1X cOmplete Protease Inhibitor Cocktail and optionally 1mM PMSF) and rotated vertically for 10 min at 4°C. Samples were centrifuged for 5 min at 1350 x g at 4°C, and resuspended in 5mL LB2 (10 mM Tris, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, with 1X cOmplete Protease Inhibitor Cocktail and optionally 1mM PMSF) and rotated vertically for 10 min at 4°C. Samples were centrifuged for 5 min at 1350 x g at 4°C, and resuspended in 1mL LB3 per 10 million cells (maximum concentration of cells for Covaris sonication), or 1 mL per ChIP. Samples were sonicated in 1mL AFA tubes for 5 min on E220 evolution Covaris with settings Peak power = 140, Duty Factor = 10, Cycles per burst = 200 to achieve chromatin sized approximately 500-2000bp. Following sonication, samples were re-combined (if aliquoted for sonication), Triton X-100 was added to the fragmented chromatin to a final concentration of 1%, and the chromatin divided for input (1-2%) and ChIP samples. 5μg anti-histone antibody or 5-9μg anti-transcription factor antibody was added per ChIP sample, and incubated overnight at 4°C.  Protein G Dynabeads (ThermoFisher) were first blocked with Block solution (0.5% BSA (w/v) in 1X PBS) and then added to cleared chromatin to bind antibody-bound chromatin for a 4-6 hour incubation. Chromatin-bound Dynabeads were washed at least 6 times with chilled RIPA wash buffer (50 mM Hepes-KOH pH 7.5, 500 mM LiCl, 1 mM EDTA, 1% NP-40, 0.7% Na-Deoxycholate), followed by a wash with chilled TE + 50 mM NaCl. Chromatin was eluted for 15-30 min in Elution Buffer (50 mM Tris, 10 mM EDTA, 1% SDS) at 65°C with frequent vortexing. The ChIP and input samples were then incubated at 65°C overnight to reverse cross-links (12-16 hours). Samples were diluted and sequentially digested with RNase A (0.2 mg/mL) for 2 hours at 37°C followed by Proteinase K (0.2 mg/mL) for 2 hours at 55°C for 2-4 hours to digest protein. ChIP and input samples were purified by phenol-chloroform-isoamylalcohol extraction and precipitation with final concentration 70% ethanol, 0.3M NaOAc pH 5.2 and 1.5μL glycogen. For library preparation, samples were quantified by Qubit dsDNA HS assay kit, and 10-30ng of ChIP DNA was used for library preparation with end repair, A-tailing, and adaptor ligation (NEB). Following USER enzyme treatment, samples were purified using Nucleospin Gel and PCR Cleanup (Takara) and separated by gel electrophoresis and size-selected for 220-500 bp by gel extraction. Libraries were then amplified to add indices using NEBNext HiFi 2X PCR mix and NEBNext Multiplex Oligos for Illumina kit (NEB, E7335S) with 9-15 cycles (as determined by qPCR). ChIP libraries were purified by two rounds of XP SPRI bead clean-up to deplete adaptors. Library concentration and quality was assessed by Bioanalyzer (to determine size) and KAPA qPCR was used to pool multiple libraries. Samples were sequenced using NextSeq or HiSeq 4000 platform (2x 75bp).
GEO Accession
GSM4314797