GSM6705564: ChIP-BS-seq_ESC_WT_H3K27me3_Input_Rep2; Mus musculus; OTHER
5 million cells were resuspended in 500 µl cell lysis buffer (20 mM Tris-HCl ph8, 85 mM KCl, 0.5% NP40) and incubated for 5 minutes on ice followed by 2500 g centrifugation at 4 ºC for 5 minutes. Supernatant was removed and pelleted nuclei were resuspended in 100 µl PBS, after which an additional 100 µl of 2x lysis buffer supplemented with 40 U/µl micrococcal nuclease (NEB) was added (100 mM Tris-HCL pH 8.0, 300 mM NaCl, 2% TritonX-100, 0.2% sodium deoxycholate, and 10 mM CaCl2). Nuclear lysis was carried out on ice for 20 minutes followed by a 25 minute incubation at 37 ºC, which was previously experimentally determined to yield mono-nucleosome sized fragments. The micrococcal nuclease reaction was terminated with the addition of 800 µl lysis dilution buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% TritonX-100, 50 mM EGTA, 50 mM EDTA, and 0.1% sodium deoxycholate) and 2 µg of H3K27me3 antibody was added (Thermo Scientific MA5-11198). Immunoprecipitation was carried out at 4 ºC overnight with gentle rotation followed by incubation with protein A dynabeads (Thermo Scientific) for 4 hours the next day. Bead bound immune complexes were washed at 4 ºC two times with RIPA buffer (0.1% DOC, 0.1% SDS, 1% Triton X-100, 10mM Tris-HCl pH 8.0, 1mM EDTA, 140 Mm NaCl), and then one time with each of the following: RIPA high salt (0.1% DOC, 0.1% SDS, 1% Triton X-100, 10mM Tris-HCl pH 8.0, 1mM EDTA, 360 mM NaCl), LiCl wash buffer (250mM LiCl, 0.5% NP40, 0.5% deoxycholate, 1mM EDTA, 10mM Tris-HCl, pH 8.0), and TE pH 8.0. DNA was then eluted from the protein A beads by dissolving them in 100 µL ChIP elution buffer (TE, 0.1% SDS, and 300mM NaCl) with 0.2 mg/mL Proteinase K (Invitrogen) and incubating at 55 ºC overnight. The next day 300 µl TE was added to the reaction along with 400 µl phenol-chloroform-isoamyl alcohol (Invitrogen), the solution was briefly vortexed, and then centrifuged at 21,000 g for 10 minutes at room temperature in phase lock tubes (VWR). After centrifugation, the aqueous phase was collected and combined with 20 µL 5M NaCl, 1 µL 20 mg/mL glycogen (Thermo Scientific), and 880 µL 100% ethanol. This solution was stored at -20 ºC overnight and then centrifuged at 21,000 g 4 ºC for 1 hour the next day. This was followed by two washes with 70% ethanol and eluted in 20 µL 1x TE. The resulting DNA was bisulfite converted with the EZ DNA methylation gold kit (Zymo) and used as input for the Accel-NGS® Methyl-Seq DNA Library Kit (Swift/IDT) following the manufacturer's protocol. All libraries were sequenced using 100 bp paired-end sequencing (200 cycles kit) on a NovaSeq 6000 platform.