Cells were cross-linked for 10 min at room temperature with 1% formaldehyde solution with gentle agitation and quenched with 0.125 M glycine. Cross-linked cells were washed once with PBS, added swelling buffer (10 mM Tris-HCl pH8.0, 1.5 mM MgCl2, 10 mM NaCl, 0.5% NP-40) and incubated on ice for 10 minutes. After centrifugation, pellets were suspended with RIPA buffer (10 mM Tris-HCl pH8.0, 1 mM EDTA, 140 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.1% Sodium Deoxycholate) containing proteinase inhibitor cocktail (Roche), and then were sonicated by using a BioRuptor sonicator (Diagenode). After centrifugation, solubilized chromatin was recovered and incubated with 5 µg of antibody bound to protein A/G magnetic beads (Pierce). After overnight incubation at 4ºC, the magnetic beads were washed with RIPA buffer twice, RIPA high salt buffer (10 mM Tris-HCl pH8.0, 1 mM EDTA, 500 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.1% Sodium Deoxycholate), LiCl buffer (10 mM Tris-HCl pH8.0, 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, 0.5% Sodium Deoxycholate) and TE buffer. The bound materials were eluted with elution buffer (10 mM Tris-HCl pH8.0, 5 mM EDTA, 300 mM NaCl, 0.1% SDS) overnight at 65ºC and treated with RNaseA for 30 min for 37°C and further incubation with Proteinase K for 1 hour at 37ºC. Then DNA was purified with a PCR purification kit (Qiagen). ChIP-seq libraries were prepared by using the NEBNext Ultra II DNA prep kit (NEB E7645) following the manufacturer's instruction and sequenced on a Next-Seq 500 Sequencer (Illumina).
GEO Accession
GSM4377993