GSM8132893: E14_ES_RA0h_rep1_nChIP.H3K27ac; Mus musculus; ChIP-Seq
For native ChIP-seq, 5 x 10^7 mouse ESCs (both untreated and following RA treatment) were pelleted and nuclei were released by resuspending in ice cold lysis buffer (10mM Tris-HCl pH 8.0, 10 mM NaCl, 3 mM MgCl2, 0.1% NP40, 5 mM N-ethylmaleimide). Nuclei were then washed, and resuspended in 1ml of MNase digestion buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 3 mM MgCl2, 0.1% NP40, 0.25M sucrose, 3mM CaCl2, 10 mM N-ethylmaleimide, 1x cOmplete protease inhibitor cocktail (Roche)). Each sample was then incubated with 200 units of MNase (ThermoFisher Scientific) at 37°C for 5 min, followed by the addition of 4 mM EDTA to halt MNase digestion. Following centrifugation at 1500 g for 5 min at 4°C, the supernatant (S1) was retained. The remaining pellet was incubated with 300 µl of nucleosome release buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 0.2 mM EDTA, 1x protease inhibitors, 10 mM N-ethylmaleimide) at 4°C for 1 h, passed five times through a 27G needle using a 1 ml syringe, and spun at 1500 g for 5 min at 4°C. The second supernatant (S2) was collected and combined with corresponding S1 sample from above. A small amount of S1/S2 DNA was purified and visualized on a 1.5% agarose gel to confirm digestion to mostly mono-nucleosomes. For a given ChIP, purified nuclease-digested chromatin from 5 × 10^6 ES cells was 10-fold diluted in native ChIP incubation buffer (10mM Tris-HCl pH 7.5, 70mM NaCl, 2mM MgCl2, 0.1% Triton X-100, 2mM EDTA, 5mM Sodium butyrate, 1× cOmplete protease inhibitor cocktail (Roche)) before being pre-cleared for 2h with Protein A Dynabeads (Life Technologies #10001D) blocked with 0.05mg/ml tRNA and 0.2mg/ml BSA. The pre-cleared chromatin was further incubated overnight at 4 °C with the appropriate antibody: anti-H3K27ac (CST, #8173, 5µg), anti-H3K27me3 (Millipore, #07-449, 5µg), anti-H3K4me1 (CST, #5326, 5µg). Antibody-bound chromatin was captured using blocked Dynabeads for 1h at 4 °C and isolated through magnetizing on ice. After four washes with ice-cold native ChIP wash buffer (20mM Tris-HCl pH 7.5, 125mM NaCl, 0.1% Triton X-100, 2mM EDTA) and one wash with ice-cold 1x TE buffer, the ChIP DNA was eluted in fresh elution buffer (1% SDS, 0.1 M NaHCO3) and incubated with shaking at 25 °C for 30min. In parallel, a matched 10% input sample was identically processed. Both ChIP and input DNA were purified using ChIP DNA Clean and Concentrator Kit (Zymo Research # D5205) following manufacturer protocol. Crosslinked ChIP-seq was performed to profile RNAP2 binding (NTD, Ser2p, Ser5p). Briefly, 5 × 10^7 ES cells were washed with 1× PBS and crosslinked with 1% formaldehyde. After 15 min incubation at RT with rotation, the fixation reaction was quenched with 0.15M glycine solution and washed again with 1x PBS. The pellet was stored at -80°C. For the nuclei isolation and sonication, the pellet was thawed on ice and then resuspended in 10mL LB1-solution (10% glycerol, 50mM HEPES-KOH (pH 7.9), 0.14 M NaCl, 1mM EDTA, 0.5 % NP-40 (IGEPAL), 0,25% Triton X100, 1x cOmplete protease inhibitor cocktail (Roche)) and rotated at 4°C for 10min. After centrifugation (5 min, 1000xg, 4°C), the supernatant was removed and the cells were resuspended in 10 mL LB2 solution (10mM Tris-HCl (pH 8.0), 0.2M NaCl, 1mM EDTA, 0.5mM EGTA, 1x cOmplete protease inhibitor cocktail (Roche)), rotated at 4°C for 5min and centrifuged (5 min, 1000xg, 4°C). The cell pellet was resuspended in 1mL LB3 (10mM Tris-HCl (pH 8.0), 0.1M NaCl, 1mM EDTA, 0.5mM EGTA, 1x cOmplete protease inhibitor cocktail (Roche)) and transferred to a sonication tube containing sonication beads (Diagenode). Sonication was performed using the Bioruptor® Pico sonicator (Diagenode) for 20 cycles, 30 sec on / off. The sonicated cell solution was transferred to 1.5 mL collection tubes and 100 µ