GSM7587801: Control_sample; Mus musculus; ChIP-Seq
Cells were fixed with 2mM DSG (Thermo Fisher Scientific, 20593) for 30 min at room temperature and replaced with fixing buffer (50 mM HEPES-NaOH (pH 7.5), 100 mM NaCl, 1 mM EDTA) containing 1% paraformaldehyde (Electron Microscopy Sciences, 15714) and crosslinked for 10 min at 37°C.Crosslinking was quenched by adding glycine to a final concentration of 0.125 M. The nuclear fraction was extracted by first resuspending the pellet in 1 ml of lysis buffer (50 mM HEPES-NaOH (pH 8.0), 140 mM NaCl, 1mM EDTA, 10% glycerol, 0.5% NP-40, and 0.25% Triton X-100) for 10 min at 4°C. Cells were pelleted, and washed in 1 ml of wash buffer (10 mM Tris-HCL (pH 8.0), 200 mM NaCl, 1 mM EDTA) for 10 min at 4°C. Cells were then pelleted and resuspended in 1 ml of shearing buffer (10 mM Tris-HCl (pH 8), 1 mM EDTA, 0.1% SDS) and sonicated.Lysate was centrifuged for 5 min at 14,000 rpm to purify the debris. Then 100 ml of 10% Triton X-100 and 30 ml of 5M NaCl were added. The sample was then incubated with 20 ml of Dynabeads Protein G (LifeTechnologies,10003D) for 1 h at 4°C. Primary antibodies were added to each tube and immunoprecipitation (IP) was conducted overnight in the cold room. Cross-linked complexes were precipitated with Dynabeads Protein G for 2 hr at 4°C. The beads were then washed in low salt wash buffer (20 mM Tris-HCl pH 8, 150 mM NaCl, 10 mM EDTA, and 1% SDS) for 5 min at 4°C, high salt wash buffer (50 mM Tris-HCl pH 8, 10 mM EDTA, and 1% SDS) for 5 min at 4°C and LiCl wash buffer (50 mM Tris-HCl pH 8, 10 mM EDTA, and 1% SDS) for 5 min at 4°C. DNA was eluted in elution buffer (100 mM sodium bicarbonate and 1% SDS). Cross-links were reversed overnight at 65°C. RNA and protein were digested with 0.2 mg/ml RNase A for 30 min at 37C followed by 0.2 mg/ml Proteinase K for 1 h at 55°C. DNA was purified with phenol-chloroform extraction and isopropanol precipitation. ChIP-seq libraries were prepared using the Rubicon ThruPLEX DNA-seq Kit from 1 ng of purified ChIP DNA or input DNA according to the manufacturer's protocol.