GSM8885830: CTCF ChIP-seq in 5KOcln55 rep2; Mus musculus; ChIP-Seq
ChIP DNA was extracted by Native ChIP protocol 5 × 10⁵ iMEFs were crosslinked in 1 mL PBS supplemented with 0.1 mL Crosslinking Buffer (100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 50 mM HEPES [pH 8.0], 11% PFA) at 25°C for 20 min. The reaction was quenched by adding 0.125 M glycine. After washing with PBS, the pellet was resuspended in 300 µL SDS lysis buffer (50 mM Tris-HCl [pH 8.0], 10 mM EDTA, 1% SDS, protease inhibitor). Chromatin was sonicated using a Picoruptor (Diagenode) with a cycle of 30 sec on/30 sec off for a total of five cycles. The lysate was then centrifuged at 15,000 rpm for 10 min at 4°C. The supernatant was diluted with 1.2 mL Chromatin Dilution Buffer (10% Tris-HCl [pH 7.5], 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 1× cOmplete EDTA-free protease inhibitor ( Roche, 11836170001), 1 mM PMSF). From the diluted chromatin solution, 100 µL was set aside for input control, and the remaining portion was incubated with 8 µg of anti-CTCF antibody (Active Motif #61311) and 10 l of Protein A/G agarose beads (Santa Cruz, sc-2003). The mixture was rotated at 4°C overnight. After rotating the solution, the beads were sequentially washed by low salt buffer (150 mM NaCl, Tris-HCl pH8.0, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), high salt buffer (500 mM NaCl, Tris-HCl pH8.0, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), LiCl buffer (25 mM LiCl, 10 mM Tris-HCl pH8.0, 1 mM EDTA, 1% NP40, 1% DOC), TE, and were suspended in Salting method buffer (20 mM Tris pH8.0, 10mM EDTA, 400 mM NaCl). Input and ChIP samples were de-crosslinked by RNaseA, SDS and Protease K treatment, and purified by QIAquick PCR purification kit (Qiagen).