GSM7566824: WT_CTCF,ChIP-nexus; Homo sapiens; ChIP-Seq
Cells were crosslinked with 1% formaldehyde and quenched with glycine. Crosslinked cells were lysed twice with ChIP buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 150 mM NaCl, 1x protease inhibitors), and then were sonicated to fragmentize DNA. After centrifugation at 12000rpm for 10 min, supernatant was transferred into new tubes and incubated with an antibody overnight at 4℃ with slow rotation. Magnetic protein A/G beads were used to enrichment targeted chromatin, and the beads were sequentially washed to remove nonspecific binding protein or DNA. The NEB end-repair module (NEB E6050S) was used to blunt the DNA ends, and Klenow exo- (NEB M0212S) to add dATP at the end of DNA. Nexus adaptors were ligated into DNA using Blunt/TA Ligase Master Mix (NEB M0367L), and Klenow exo- (NEB M0212S) and T4 DNA polymerase (NEB M0203S) were sequentially used to generate blunt-end DNA. The blunt-end DNA was digested with Lambda Exonuclease (NEB M0262S) and RecJf exonuclease (NEB M0264S). After reverse-crosslinking at 65℃for overnight, the DNA fragment were extracted using phenol-chloroform and precipitated with ethanol contains sodium acetate. DNA fragment were denatured to generate ssDNA and then cyclized with ssDNA Ligase (Epicentre CL4111K). The circular DNA were annealed with oligonucleotides contain BamHI endonuclease recognition sites. After digestion with BamHI and re-precipitation using ethanol, the ChIP-nexus library was constructed with PCR.