Instrument: NextSeq 500
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: PAIRED
Construction protocol: For RING1B and SUZ12 cChIP-seq, 5x107 mouse ESCs were first crosslinked with 2 mM disuccinimidyl glutarate (DSG, Thermo Scientific) while rotating for 45 min at 25°C, and then with 1% formaldehyde (methanol-free, Thermo Scientific) for further 15 min. Reactions were quenched by addition of 125 mM glycine. Mouse ESCs were then mixed with 2x106 double-crosslinked HEK293T cells and incubated in lysis buffer (50 mM HEPES pH 7.9, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, 1x PIC) for 10 min at 4°C. The released nuclei were washed (10 mM Tris-HCl pH 8, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1x PIC) for 5 min 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% sodium deoxycholate, 0.5% N-lauroylsarcosine, 1x PIC) and sonicated for 30 min using the BioRuptor Pico (Diagenode). Following sonication, Triton X-100 was added to a final concentration of 1%. The supernatant was collected after centrifugation at 20,000 g for 10 min at 4°C. For ChIP, chromatin was diluted 10-fold in ChIP dilution buffer (1% Triton X-100, 1 mM EDTA, 20 mM Tris-HCl pH 8, 150 mM NaCl, 1x PIC) and pre-cleared for 1 h with either protein A agarose beads (Repligen, for RING1B ChIP) or protein A magnetic Dynabeads (Invitrogen, for SUZ12 ChIP) blocked with 1 mg/mL bovine serum albumin (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, either anti-RING1B (CST, D22F2, 3 μl), or anti-SUZ12 (CST, D39F6, 3 μl). Antibody-bound chromatin was captured using blocked protein A beads (agarose for RING1B, magnetic Dynabeads for SUZ12) for at least 2 h at 4°C and collected by centrifugation/on a magnetic rack. ChIP washes were performed as described previously (Farcas et al. 2013). ChIP DNA was eluted in elution buffer (1% SDS, 0.1 M NaHCO3) and cross-linking was reversed overnight at 65°C with 200 mM NaCl and 2 μL RNase A (Sigma). A matched input sample (1/10 of original ChIP reaction) was treated identically. The following day, ChIP samples and inputs were incubated with Proteinase K (Sigma) for 1.5 h at 56°C and purified using ChIP DNA Clean and Concentrator Kit (Zymo Research). For Pol II cChIP-seq, 5x107 mouse ESCs were crosslinked in 1% formaldehyde for 10 min at 25°C and then quenched with 125 mM glycine. Mouse ECSs were mixed with 2x106 single-crosslinked HEK293T cells and incubated in FA-lysis buffer (50 mM HEPES pH 7.9, 150 mM NaCl, 2 mM EDTA, 0.5 mM EGTA, 0.5% NP-40, 0.1% sodium deoxycholate, 0.1% SDS, 10 mM NaF, 1 mM AEBSF, 1x PIC) for 10 min. Chromatin was sonicated for 30 min using the BioRuptor Pico, centrifuged at 20,000 g for 10 min at 4°C, and supernatant taken as chromatin. For ChIP, 300 ug of chromatin was diluted to 1 ml in FA-lysis buffer and pre-cleared for 1 h with protein A agarose beads blocked with 1 mg/mL BSA and 1 mg/mL yeast tRNA. For each ChIP reaction, diluted and pre-cleared chromatin was incubated overnight with the appropriate antibody, anti-Rbp1-NTD (CST, D8L4Y, 15 μl) to detect total Pol II, anti-Rbp1-CTD-Ser5P (CST, D9N5I, 12.5 μl), or anti-Rbp1-CTD-Ser2P (CST, E1Z3G, 12.5 μl). Antibody-bound chromatin was isolated using blocked protein A agarose beads for 3 h at 4°C. 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, de-crosslinked and purified as described above, along with the matched input samples. For H2AK119ub1, H3K27me3 and H3K4me3 cChIP-seq, 5x107 mouse ESCs were mixed with 2x107 Drosophila SG4 cells in PBS. The 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% NP-40, 5 mM NEM). Nuclei were then washed, and resuspended in 1 mL of MNase digestion buffer (10 mM Tris-HCl pH 8, 10 mM NaCl, 3 mM MgCl2, 0.25 M sucrose, 3 mM CaCl2, 10 mM NEM, 1x PIC). Samples were digested with 150 units of MNase (Fermentas) for 5 min at 37°C, stopped by 4 mM EDTA. The supernatant (S1) was collected following centrifugation at 1500 g for 5 min at 4°C. 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 NEM, 1x PIC) at 4°C for 1 h, passed five times through a 27G needle, and spun at 1500 g for 5 min at 4°C. The second supernatant (S2) was collected and combined with corresponding S1 supernatant, and stored at -80°C until use. Digestion to predominantly mono-nucleosomal fragments was confirmed by agarose gel electrophoresies of purified DNA. Native chromatin was diluted 10-fold in native ChIP incubation buffer (70 mM NaCl, 10 mM Tris–HCl pH 7.5, 2 mM MgCl2, 2 mM EDTA, 0.1% TritonX-100, 10 mM NEM, 1x PIC). For each ChIP reaction, 1 ml of diluted nucleosomes was incubated overnight at 4°C with the appropriate antibody, anti-H2AK119ub1 (CST, D27C4, 5 μL), anti-H3K27me3 (in-house, 5 μL) or anti-H3K4me3 (in-house, 3 μL). Antibody-bound nucleosomes were captured by incubation for 1 h at 4°C with protein A agarose beads, pre-blocked overnight in native ChIP incubation buffer supplemented with 1 mg/ml BSA and 1 mg/ml yeast tRNA. The beads were then washed four times with native ChIP wash buffer (20 mM Tris–HCl pH 7.5, 2 mM EDTA, 125 mM NaCl, 0.1% TritonX-100) and once with TE buffer pH 8. Immunoprecipitated DNA was eluted using 100 μl of elution buffer (1% SDS, 0.1 M NaHCO3) and purified using ChIP DNA Clean and Concentrator Kit. DNA from a matched input sample (corresponding to 10% of the original ChIP reaction) purified in the same way. cChIP-seq libraries for both ChIP and input samples were prepared using NEBNext Ultra DNA Library Prep Kit for Illumina (double-crosslinked and native ChIP-seq) or NEBNext Ultra II DNA Library Prep Kit for Illumina (Pol II ChIP-seq), following manufacturer's guidelines. Samples were indexed using NEBNext Multiplex Oligos. The average size and concentration of all libraries was analysed using the 2100 Bioanalyzer High Sensitivity DNA Kit (Agilent) followed by qPCR using SensiMix SYBR (Bioline, UK) and KAPA Illumina DNA standards (Roche). Libraries were sequenced as 40 bp paired-end reads on the Illumina NextSeq 500.