GSM8477296: E14_ES_RA4h_rep1_xChIP.Ser5p; 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 µL of 10% Triton X100 in LB3 was added and mixed by inversion. After centrifugation at 20,000 xg for 10 min at 4°C, the supernatant was aliquoted into dry ice-cooled tubes (150µg chromatin/tube). Sonication was confirmed with a 1.5 % agarose gel. For each ChIP, either 150 µg (NTD polymerase) or 300 µg (Ser2p, Ser5p) chromatin were diluted 10-fold in ChIP dilution buffer (10% Triton X100, 1mM EDTA, 20mM Tris-HCl (pH 8.0), 0.15M NaCl) and then pre-cleared for 2h with Protein A Dynabeads (Life Technologies #10001D) blocked with 0.05mg/ml tRNA and 0.2mg/ml BSA. Prior to the addition of the antibody, 2% input was taken from the pre-cleared chromatin. The pre-cleared chromatin was further incubated overnight at 4 °C with the appropriate antibody: anti-Rpb1 NTD (CST, #14958, 7.5µg/150µg chromatin), anti-phospho-Rpb1 CTD (Ser2p) (CST, #13499, 6.25µg/150µg chromatin), anti-phospho-Rpb1 CTD (Ser5p) (CST, #13523, 6.25µg/150µg chromatin). Antibody-bound chromatin was captured with blocked Dynabeads for 1h at 4 °C and isolated by magnetisation on ice. Dynabeads were washed with low salt buffer (0.1% SDS, 1% Triton X100, 2mM EDTA, 20mM Tris-HCl (pH 8.0), 0.15M NaCl), high salt buffer (0.1% SDS, 1% Triton X100, 2mM EDTA, 20mM Tris-HCl (pH 8.0), 0.5M NaCl), LiCl buffer (0.25M LiCl, 0.1% NP40, 0.1% sodium deoxycholate, 1mM EDTA, 10mM Tris-HCl (pH 8.0)) and twice with TE buffer (10mM Tris-HCl (pH 8.0), 0.1mM EDTA). The chromatin-bound beads were eluted with 100µl/150µg chromatin elution buffer (1% SDS, 0.1M NaHCO3) and incubated at 25 °C for 30min with shaking. The input sample was topped up to the same volume as the ChIP sample and 4µL 4M NaCl and 2µL RNaseA (Qiagen #19101, 20mg/mL) were added. Samples were incubated overnight at 65°C with slow shaking. Then 1µL of Proteinase K was added and the samples were incubated at 55°C for 1 hour with slow shaking. Both ChIP and input DNA were purified using the ChIP DNA Clean and Concentrator Kit (Zymo Research # D5205) following manufacturer protocol. Libraries for both ChIP and Input samples were prepared using NEBNext Ultra II DNA Library Prep Kit for Illumina, following manufacturer's guidelines. Samples were indexed using NEBNext Multiplex Oligos. The average size of all libraries was analysed using either the 2100 Bioanalyzer High Sensitivity DNA Kit (Agilent, nChIP) or the TapeStation (xChIP). Library concentration was further determined either by using qPCR with SensiMix SYBR (Bioline, UK)  and KAPA Illumina DNA standards (Roche) for nChIP or by quantification via Qubit for xChIP. Libraries were sequenced as 40-42 bp paired-end reads on Illumina NextSeq 500 platform in biological triplicates or quadruplicates.