GSM6128373: Control, ONECUT3-FLAG, rep1; Mus musculus; ChIP-Seq
MEF cells were crosslinked with 1% formaldehyde at room temperature for 10 minutes. 1.5 M Glycine solution was used to terminate the fixation reaction. After washing with the iced PBS and homogenizing, cells were then lysed with SDS cell-lysis buffer (50 mM Tris pH 8.0, 1 mM EDTA pH 8.0, 0.1% wt/vol SDS and 1 mM PMSF) for 10 min on ice, and the chromatin was sonicated to 100–500bp in a Bioruptor (Diagenode). The size of the sheared chromatin was verified by agarose gel electrophoresis after reverse cross-linking.     For immunoprecipitation, Dynabeads Protein G (Thermo, 10003D) was pre-washed was 0.5% BSA and conjugated to 5 μg of ChIP antibody or normal IgG at 4 °C for 4-6h. After washing with the RIPA/150mM NaCl washing buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1 mM EDTA pH 8.0, 1% wt/vol sodium deoxycholate, 1% vol/vol Triton X-100, 0.1% wt/vol SDS and 1 mM PMSF), the conjugated beads were added to the chromatin slurry and incubated at 4 °C for 12 hours. Afterward, beads were washed twice with RIPA/150mM NaCl washing buffer, twice with RIPA/500mM NaCl washing buffer (50 mM Tris pH 8.0, 500 mM NaCl, 1 mM EDTA pH 8.0, 1% wt/vol sodium deoxycholate, 1% vol/vol Triton X-100, 0.1% wt/vol SDS and 1 mM PMSF), four times LiCl washing buffer (10 mM Tris pH 8.0, 250 mM LiCl, 1 mM EDTA pH 8.0, 0.5% vol/vol NP-40 and 0.5% vol/vol Sodium Deoxycholate) and twice with TE buffer (10 mM Tris pH 8.0, 300 mM NaCl and 0.5 mM EDTA pH 8.0). The precipitated beads were diluted with ChIP elution buffer (10 mM Tris pH 8.0, 250 mM LiCl, 1 mM EDTA pH 8.0, 0.5% vol/vol NP-40 and 0.5% wt/vol SDS). One forth of the solution with beads were added with SDS protein lysis buffer for WB to verify the specificity of ChIP. The rest of the ChIP'd beads and the input were reversed cross-linking with 5 M NaCl solution at 65 °C overnight.     The procedure of the DNA recovery from the immunoprecipitation is as follows. The above reversed cross-linking solution was mixed with 2x SDS lysis buffer (200 mM Tris pH 8.0, 1 M NaCl, 100mM EDTA pH 8.0 and 0.5% wt/vol SDS) with Proteinase K at 200x rpm for 15 minutes and followed by RNase A at 37 °C for 30 minutes. 3 M NaAc (pH 5.2) was used to remove the protein. The clean supernatant was mixed with Isopropanol and Glycogen to precipitate the DNA. After washed twice with pre-cooled 75% ethanol, DNA pellets were dried for 10 minutes, dissolved in 10 mM Tris-HCl (pH 8.0) and then measured by the Qubit dsDNA HS Assay Kit (Thermo Fisher) on the Qubit instrument (ThermoFisher Scientific). NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, E7645)  was used for library preparation.