Instrument: NextSeq 500
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: PAIRED
Construction protocol: For RING1B and SUZ12 cChIP-seq, 5 x 10^7 mouse ESCs (both untreated and following 72 hours tamoxifen treatment) were mixed with 2 x 10^6 human HEK293T cells. Cells were resuspended in 10 ml phosphate buffered saline (PBS) and crosslinked at 25°C firstly with 2 mM DSG (Thermo Scientific) for 45 mins, and then with 1% formaldehyde (methanol-free, Thermo Scientific) for a further 15 minutes. Reactions were quenched by the addition of 125 mM glycine. Crosslinked cells were 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) for 10 min at 4˚C. The released nuclei were then washed (10 mM Tris-HCl pH 8, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA) 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% Na deoxycholate, 0.5% N-lauroylsarcosine) and sonicated for 30 min using a BioRuptor Pico sonicator (Diagenode), shearing genomic DNA to an average size of approximately 0.5 kb. Following sonication, TritonX-100 was added to a final concentration of 1%. For ChIP, sonicated chromatin was diluted 10-fold in ChIP dilution buffer (1% Triton-X100, 1 mM EDTA, 20 mM Tris-HCl pH 8, 150 mM NaCl) and pre-cleared for 1 hour using Protein A agarose beads (Repligen) blocked with 1 mg/ml BSA and 1 mg/ml yeast tRNA. For each ChIP reaction, 1ml of diluted and pre-cleared chromatin was incubated overnight with 3 ul of the appropriate antibody, anti-RING1B (Cell Signaling Technology , D22F2) or anti-SUZ12 (CST, D39F6). Antibody-bound chromatin was captured using blocked protein A agarose for 2 hours at 4°C and collected by centrifugation. 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-links were reversed overnight at 65°C with 200 mM NaCl and 2 µl of RNase A (Sigma). A matched input sample (corresponding to 10% of original ChIP reaction) was identically treated. The following day ChIP samples and Inputs were incubated with Proteinase K (Sigma) for 1.5 hours at 56°C and purified using ChIP DNA Clean and Concentrator Kit (Zymo Research). For H2AK119ub1 and H3K27me3 native cChIP-seq, 5 x 10^7 mouse ESCs (both untreated and following 72 hours tamoxifen treatment) were mixed with 2 x 10^7 Drosophila SG4 cells in PBS. Mixed cells 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 1 ml 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 protease inhibitors (Sigma)). Each sample was then 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, 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. Libraries for both ChIP and Input samples were prepared using NEBNext Ultra 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 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 Illumina NextSeq 500 platform in biological triplicate unless otherwise specified. For allele-specific analysis in 129S1 x CAST hybrid ESC line with inducible fill-length Xist transgene, native cChIP-seq libraries were sequenced as 80 bp paired-end reads on Illumina NextSeq 500 platform to increase the number of reads overlapping allele-specific SNPs.