GSM6166235: ChIPseq-Rif1-CKO-H2A119ub; Mus musculus; ChIP-Seq
Briefly, mESCs of 80–90% confluency were crosslinked with a final concentration of 1% formaldehyde for 10 min at room temperature, and then quenched by the addition of 125 mM glycine. The cells were then rinsed twice with ice-cold DPBS, and harvested by scraping using silicon scraper. The supernatant was discarded after spinning at 1350 g for 5 min at 4 ℃, and the pellet was lysed with lysis buffer A (50 mM HEPES-KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 0.5% NP-40, 0.25% Triton X-100, 10% glycerol and protease inhibitor cocktail), incubated at 4℃ for 10 min with rotating, and collected by spinning at 1350 g for 5 min at 4℃. Cells were then resuspended in lysis buffer B (10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, and protease inhibitor cocktail), and incubated at room temperature for 10 min. The nuclei were then pelleted by spinning at 1350 g for 5 min at 4℃. The pellet was resuspended with lysis buffer C (10 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine, and protease inhibitor cocktail), incubated for 15 min on ice, and transferred into Covaris microTUBEs. The DNA was sonicated to 200 bp fragments using Covaris S220 (duty cycle: 10%; intensity: 5; cycles/burst: 200; duration: 195s). After sonication, a final concentration of 1% Triton X-100 was added and gently mixed by pipetting. The chromatin solution was clarified by spinning at 12000 rpm at 4℃ for 10 min. 50 μL of supernatant from each sample was reserved as input and the rest chromatin solution was incubated with the indicated primary antibodies overnight at 4℃. The magnetic beads (Thermo, 88803) were added after blocking in the block buffer (0.5% (w/v) RNase/DNase-free BSA in 1 × PBS), and incubated with chromatin solution and antibodies for 4 h at 4℃. The immunoprecipitant was then washed 5 times with wash buffer (50 mM HEPES-KOH, pH 7.6, 500 mM LiCl, 1 mM EDTA, 1% NP-40, 0.7% Na-deoxycholate). The DNA was then eluted with Elution Buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% SDS). To reverse the crosslinks, samples were incubated at 65℃ overnight. The RNA was digested with a final concentration of 1 mg/mL RNases A at 37℃ for 1 h, and the proteins were degraded subsequently with a final concentration of 0.5 mg/mL Proteinase K at 55℃ for 2 h. The immunoprecipitated DNA was then extracted with AMPure beads according to the manufacturer's instructions (Beckman, A63881). For ChIP-seq, 1 ng precipitated DNA or input was used to generate the DNA library using NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, E7645S).