Instrument: NextSeq 500
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: PAIRED
Construction protocol: For RING1B and SUZ12 cChIP-seq, 5×107 mouse ESCs (untreated and OHT-treated) were crosslinked in 10 ml 1x PBS with 2 mM DSG (Thermo Scientific) for 45 minutes at 25°C, and then with 1% formaldehyde (methanol-free, Thermo Scientific) for a further 15 minutes. Crosslinking was stopped by quenching with 125 mM glycine. Crosslinked ESCs were mixed with 2×106 human HEK293T cells, which have been similarly double-crosslinked, and incubated in lysis buffer (50 mM HEPES pH 7.9, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP40, 0.25% Triton-X100, 1x PIC (Roche)) for 10 minutes at 4˚C. Released nuclei were washed (10 mM Tris-HCl pH 8, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1x PIC (Roche)) for 5 minutes at 4˚C. Chromatin was then resuspended in 1 ml of sonication buffer (10 mM Tris-HCl pH 8, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na deoxycholate, 0.5% N-lauroylsarcosine, 1x PIC (Roche)) and sonicated for 30 minutes using the BioRuptor Pico (Diagenode), shearing genomic DNA to an average size of 0.5 kb. Following sonication, TritonX-100 was added to a final concentration of 1%, followed by centrifugation at 20,000 g for 10 min at 4°C to collect the clear supernatant fraction. For cChIP-seq for Pol II and its phosphorylated forms, 5×107 ESCs (untreated and OHT-treated) were crosslinked in 10 ml 1x PBS with 1% formaldehyde (methanol-free, Thermo Scientific) for 10 min at 25°C and then quenched by addition of 125 mM glycine. Cross-linked ESCs were mixed with 2×106 human HEK293T cells, which have been similarly single-crosslinked, and then incubated in FA-lysis buffer (50 mM HEPES pH 7.9, 150 mM NaCl, 2 mM EDTA, 0.5 mM EGTA, 0.5% NP40, 0.1% sodium deoxycholate, 0.1% SDS, 10 mM NaF, 1 mM AEBSF, 1×PIC) for 10 min at 4˚C. Chromatin was sonicated for 30 min using the BioRuptor Pico (Diagenode), followed by centrifugation at 20,000 g for 10 min at 4°C to collect the clear supernatant fraction. For RING1B and SUZ12 ChIP, sonicated chromatin was diluted 10-fold with ChIP dilution buffer (1% Triton-X100, 1 mM EDTA, 20 mM Tris-HCl pH 8, 150 mM NaCl, 1xPIC). For Pol II ChIP, 300 ug of chromatin per one IP was diluted in FA-lysis buffer up to a final volume of 1 ml. Diluted chromatin was pre-cleared for 1 hr using Protein A agarose beads (Repligen) that were pre-blocked with 1 mg/ml BSA and 1 mg/ml yeast tRNA. For each ChIP reaction, 1 ml of diluted and pre-cleared chromatin was incubated overnight with the appropriate antibody, anti-RING1B (CST, D22F2, 3 ul), anti-SUZ12 (CST, D39F6, 3 ul), anti-Rpb1-NTD (CST, D8L4Y, 15 μl) as a measure of total Pol II levels, anti-Rpb1-CTD-Ser5P (CST, D9N5I, 12.5 μl) and anti-Rpb1-CTD-Ser2P (CST, E1Z3G, 12.5 μl) as a measure of Pol II phosphorylation levels. To capture antibody-bound chromatin, ChIP reactions were incubated with pre-blocked protein A agarose beads (Repligen) for 2 hr (RING1B and SUZ12) or 3 hr (Pol II) at 4°C. For RING1B and SUZ12, ChIP washes were performed as described previously (Farcas et al. 2012). For Pol II, washes were performed with FA-Lysis buffer, FA-Lysis buffer containing 500 mM NaCl, DOC buffer (250 mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate, 2 mM EDTA, 10 mM Tris-HCl pH 8), followed by two washes with TE buffer (pH 8). ChIP DNA was eluted in elution buffer (1% SDS, 0.1 M NaHCO3) and cross-linking was reversed overnight at 65oC with 200 mM NaCl and 2 ul RNase A (Sigma). Matched input samples (10% of original ChIP reaction) were treated identically. The following day, ChIP samples and inputs were incubated with Proteinase K (Sigma) for at least 1.5 hr at 56°C and purified using ChIP DNA Clean and Concentrator Kit (Zymo Research). For native cChIP-seq for H2AK119ub1, H3K27me3, H3K27ac, H3K4me3, and H3K4me1, 5×107 mouse ESCs (untreated and OHT-treated) were mixed with 2×107 Drosophila SG4 cells in 1x PBS. Mixed cells were pelleted and nuclei were released by resuspending in ice-cold lysis buffer (10 mM Tris-HCl pH 8, 10 mM NaCl, 3 mM MgCl2, 0.1% NP40, 5 mM sodium butyrate, and 5 mM N-ethylmaleimide). Nuclei were then washed and resuspended in 1 ml of MNase digestion buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 3 mM MgCl2, 0.1% NP40, 0.25 M sucrose, 3 mM CaCl2, 10 mM sodium butyrate, 10 mM N-ethylmaleimide, and 1x PIC (Roche)). Each sample was incubated with 200 units of MNase (Fermentas) 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, 10 mM sodium butyrate, 10 mM N-ethylmaleimide, and 1x PIC (Roche)) at 4°C for 1 hr, 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 the corresponding S1 sample from above. Digestion to mostly mono-nucleosomes was confirmed by agarose gel electrophoresis of purified S1/S2 DNA. For ChIP, S1/S2 nucleosomes were diluted 10-fold in Native ChIP incubation buffer (70 mM NaCl, 10 mM Tris pH 7.5, 2 mM MgCl2, 2 mM EDTA, 0.1% TritonX-100, 10 mM sodium butyrate (for H3K27ac and H3K4me3 ChIPs), 10 mM N-ethylmaleimide, and 1xPIC (Roche)). Each ChIP reaction, 1 ml of diluted nucleosomes was incubated overnight at 4oC with the appropriate antibody, anti-H2AK119ub1 (CST, D27C4, 5 ul), anti-H3K27me3 (in-house, 5 ul), anti-H3K27ac (CST, D5E4, 3 μl), anti-H3K4me3 (in-house, 4 ul) or anti-H3K4me1 (CST, D1A9, 5 ul). Antibody-bound nucleosomes were captured for 1 hr at 4°C using protein A agarose (Repligen) beads, pre-blocked in Native ChIP incubation buffer supplemented with 1 mg/ml BSA and 1 mg/ml yeast tRNA, and collected by centrifugation. Immunoprecipitated material was washed four times with Native ChIP wash buffer (20 mM Tris pH 7.5, 2 mM EDTA, 125 mM NaCl, 0.1% Triton-X100) and once with TE buffer (pH 8). ChIP DNA was eluted using 100 ul of elution buffer (1% SDS, 0.1 M NaHCO3) for 30 min at room temperature, and then purified using ChIP DNA Clean and Concentrator Kit (Zymo Research). For each ChIP sample, DNA from a matched input control (corresponding to 10% of original ChIP reaction) was purified in the same way. cChIP-seq 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 and concentration of all libraries were analysed using the 2100 Bioanalyzer High Sensitivity DNA Kit (Agilent) followed by qPCR quantification using SensiMix SYBR (Bioline, UK) and KAPA Illumina DNA standards (Roche). Libraries were sequenced as 40 bp paired-end reads in biological triplicate or quadruplicate on Illumina NextSeq 500 platform.