title: GSM7866215: hESC cells, WT, CTCF, ChIP rep1; Homo sapiens; ChIP-Seq
library_construction_protocol: Cells cultured in 10cm dishes were crosslinked in 1% formaldehyde for 15 minutes at room temperature. Crosslinking was quenched with 125 mM glycine. The cells were precipitated with centrifugation at 1000 r.p.m. for 10 minutes and washed with PBS. The pellet was lysed in ChIP buffer 1 (50mM HEPES (Abcam), 140mM NaCl, 1mM EDTA (Invitrogen), 10% Glycerol (Solarbio), 0.5% Igepal (Sigma), 0.5% Triton X-100 and Phenylmethylsulfonyl fluoride (PMSF, Roche)), rotated at 4℃ for 30 minutes and centrifuged at 800g for 10 minutes. The pellet was resuspended in ChIP buffer 2 (200mM NaCl, 1mM EDTA, 0.5mM EGTA, 10mM Tris-HCl (PH 8.0) and PMSF), rotated at 4℃ for 30 minutes and centrifuged at 800g for 10 minutes. Then 200 µl sonication buffer (50mM HEPES, 140mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate (Shanghai Chemical Reagent Factory), 0.3% SDS (Invitrogen) and 1×protease inhibitor cocktail (APE×BIO)) was used to resuspend the pellet and the mixture was sonicated for 13 cycles. The chromatin solution was centrifuged at 4℃ in 10000g for 10 minutes to transfer the supernatant to a new centrifuge tube. 10% of the chromatin solution was saved as an input control, half of the remaining solution was incubated with CTCF antibody (Abcam) and half was incubated with IgG antibody (Abcam). The chromatin solution was rotated at 4℃ overnight with corresponding antibody and protein A/G beads (ThermoFisher) . The next day the sample was taken out and placed on the magnetic adsorption rack (DynaMag™-2 magnet, Thermo Fisher) to remove the supernatant. Then the following four solutions are used successively to clean the chromatin-beads complexes, low-salt solution (0.1% SDS, 1% Triton-X 100, 2mM EDTA, 20mM Tris-HCl (PH 8.0), 150mM NaCl), high-salt solution (0.1% SDS, 1% Triton-X 100, 2mM EDTA, 20mM Tris-HCl (PH 8.0), 500mM NaCl), LiCl solution (250mM LiCl (Sigma), 1% NP40 (ThermoFisher), 1mM EDTA, 10mM Tris-HCl (PH 8.0), 1% sodium deoxycholate), TE buffer (10mM Tris-HCl (PH 8.0), 1mM EDTA). The cleaned sample was eluted with solution (1% SDS, 0.1M NaHCO3 (Sinopharm Chemical Reagent Co.,Ltd.), 0.1mg/ml RNaseA (NEB)) at 37℃ in 1200 r.p.m. for 30 minutes on shaker. Then protease K (1mg/ml, Promega) was added to the input, IP and IgG groups respectively followed by digestion at 50℃ for 45 minutes. DNA was extracted with phenol–chloroform, precipitated with 1/10 volume of 3M sodium acetate (ThermoFisher) and 2.5 volumes of 100% ethanol, dissolved in ddH2O, and stored at -20℃. extracted DNA was first interrupted by ultrasound into 150-500 bp. After end repair and A-Tailing, PE adapter was ligated.After 2% agarose fragment screening, PCR amplification to obtain the ChIP final library.