GSM4979836: R1-CT-H3K27me3, 1; Mus musculus; ChIP-Seq
Cells were cross-linked in 1% formaldehyde (28908, Thermo Fisher Scientific) for 10 min at RT with intermittent agitation every 2 min. After fixation, cells were washed 3 times with ice-cold PBS and scraped in PBS with 1× protease inhibitor cocktail mix (05056489001, Sigma-Aldrich). Cells were collected by centrifugation (8 min, 1500 rpm, 4°C) and resuspended (108 cells) in 50 ml of lysis buffer (50 mM HEPES [pH 7.5], 140 mM NaCl, 0.1% Na-deoxycholate, 1 mM EDTA, 0.1% SDS, 1% NP-40 [IPEGAL], 1X protease inhibitor cocktail mix) for 30 min on ice. Nuclei were collected by brief centrifugation (2,500 rpm, 10 min, 4°C) and resuspended (nuclei/10M cells) in 300 µl of sonication buffer (10 mM Tris-HCl [pH 8.0], 100 mM NaCl, 1 mM EDTA, 1% SDS, and 1X protease inhibitor cocktail mix) and sonicated using a Diagenode bioruptor to generate 200- to 1,000-bp fragments, as determined by agarose gel electrophoresis. 30 µl magnetic beads (Protein A, 10002D, Life technologies) slurry was pre-washed three times with 1ml of PBS (1% BSA) before antibody incubation. Beads were collected and resuspended in 300ul of PBS (1% BSA). 5μl of H3K27me3 antibody (17-622; Merck-Millipore), 2 µl of H3K4me3 antibody (ab8580; Abcam) and 5 µl of H3K27ac antibody (ab4729; Abcam) were added for each ChIP and incubated for 4 hours at 4°C. Antibody coupled magnetic beads were washed three times with PBS (1% BSA), and 300 µl of sonicated cell lysates were diluted 6 fold in binding buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl [pH 8.0]). The diluted chromatin was incubated over night with antibody coupled magnetic beads at 4°C. The immunoprecipitates were washed 5 times with RIPA buffer (50 mM HEPES [pH 7.6], 1 mM EDTA, 0.7% Na-deoxycholate, 1% NP-40, 0.5M LiCl) and twice with TE buffer (10 mM Tris-HCl [pH 8.0], 0.1 mM EDTA). The DNA was recovered by reversing the cross-links in 1% SDS, 0.1 M NaHCO3 for 6 h at 65°C, purified using ChIP DNA clean and concentrator kit (Ozyme/Cell Signaling Technology) and eluted in 30 µl of elution buffer provided with the kit. Eluted DNA was quantified by using Qubit ds DNA high sensitivity kit (Life Technologies) and treated with RNase A at 37°C for 1hour. Two columns were combined to get the desired amount of DNA for library preparation (30 ng) for respective histone marks and experiments were done in duplicates for each histone mark. For the spike-in, 2 µl of spike-in-antibody (61686, Active motif) was added with the respective anti-histone antibodies during antibody incubation with the beads and 7 µl of spike-in chromatin (53083, Active motif) was added with the sonicated chromatin (300µl) for each CHIP experiment. The libraries were prepared by MGX Sequencing platform (Montpellier, France) using TruSeq CHIP library preparation kit (Illumina) following guidelines and sequenced using HiSeq-2500 apparatus.