GSM5057347: RNF20-WT_IgG_1; Mus musculus; ChIP-Seq
Approximately 1×106 cells were crosslinked with 1% formaldehyde for 10 min at room  temperature. Fixation was stopped by adding Glycine at a final concentration of 125 mM and  incubated for 15 min at room temperature. After washing twice with PBS, cells were resuspended  in cell lysis buffer (5 mM PIPES, pH 8.0, 85 mM KCl, 0.5% NP-40 and 1 × protease inhibitor  cocktail) and incubate for 10 min on ice. The cell nuclei were pelleted at 1000 rpm for 10 min at  4°C, and the nuclear pellet was resuspended in nuclei lysis buffer (50 mM Tris-HCl, pH 8.0, 10  mM EDTA, 1% SDS and 1 × protease inhibitor cocktail) and incubate for 10 min on ice.  Chromatin DNA was sheared to 250-600 bp by sonication. 5% chromatin fragments were saved  as input and the remaining were incubated with magnetic beads conjugated with antibody  overnight at 4°C. Beads were sequentially washed four times with high-salt wash buffer (50 mM  HEPES, pH 7.9, 500 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100, 0.1% deoxycholate  and 1 × protease inhibitor cocktail), and two times with TE buffer (10 mM Tris-HCl, pH 8.0 and  1 mM EDTA). The protein-chromatin complex was eluted with elution buffer (50 mM Tris-HCl,  pH 8.0, 10 mM EDTA, 1% SDS with 1 μl of proteinase K (20 μg/μl)) for 2 h at 55°C and decrosslinked by incubation overnight at 65°C. The precipitated hybrid fragment was cleaned by  phenol:chloroform:isoamyl alcohol once, followed by ethanol precipitation. Sequencing libraries were generated with NEBNext® UltraTM II DNA Library Prep Kit for Illumina (E7645S), according to the manufacturer's instructions. Briefly, 10 ng of ChIP DNA were used for library preparation. NEBNext Multiplex Oligos for Illumina (Set 1, NEB #E7600) were used for PCR amplification of adaptor-ligated DNA. Libraries were purified with SPRIselect® Reagent Kit (Beckman Coulter, Inc. #B23317). Sequencing was performed on Illumina NovaSeq 6000 System.