Instrument: NextSeq 500
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: PAIRED
Construction protocol: For cross-linked ChIP, 50x10^6 ES cells were fixed for 45min with with 2mM DSG (ThermoFisher scientific) in PBS and 12.5 min with 1% formaldehyde (methanol-free, ThermoFisher scientific). Reactions were quenched by the addition of glycine to a final concentration of 125 µM and the fixed cells were washed in ice-cold 1xPBS and snap frozen in liquid nitrogen. 50x10^6 HEK293T cells were fixed as above, snap frozen in 2x106 aliquots, and stored at -80°C until further use. For calibrated ChIPseq, 2x10^6 HEK293T cells were resuspended in 1ml ice-cold lysis buffer (50mM HEPES pH 7.9, 140mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP40, 0.25% Triton X-100) and added to 50x10^6 fixed ESCs, resuspended in 9ml lysis buffer. The cell suspension was incubated for 10 min at 4°C. The released nuclei were then washed (10mM Tris-HCl pH 8, 200mM NaCl, 1mM EDTA, 0.5mM EGTA) for 10 min at 4°C. The chromatin pellet was resuspended in 1mL of ice-cold sonication buffer (10mM Tris-HCl pH 8, 100mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.1% Na deoxycholate, 0.5% N-lauroylsarcosine) and sonicated for 25 min using a BioRuptor Pico sonicator (Diagenode), shearing genomic DNA to produce fragments between 300bp and 1kb. Following sonication, Triton X-100 was added to a final concentration of 1%. Two hundred and fifty µg chromatin diluted 10-fold in ChIP dilution buffer (1% Triton-X100, 1mM EDTA, 20mM Tris-HCl (pH 8.0), 150mM NaCl) was used per IP. Three reactions were set up per condition to allow for maximal DNA recovery suitable for library preparation. Chromatin was precleared with protein A Dynabeads blocked with 0.2 mg/ml BSA and 50 µg/ml yeast tRNA and incubated with the respective antibodies overnight at 4°C. Antibody-bound chromatin was purified using blocked protein A Dynabeads for 3 hours at 4°C. ChIP washes were performed as described in Farcas et al 2012. ChIP DNA was eluted in ChIP elution buffer (1% SDS, 100mM NaHCO3) and reversed cross-linked overnight at 65°C with 200mM NaCl and RNase A (Sigma). The reverse cross-linked samples were treated with 20 μg/ml Proteinase K and purified using ChIP DNA Clean and Concentrator kit (Zymo research). The three reactions per condition were pooled at this stage. For each sample, corresponding Input DNA was also reverse cross-linked and purified. The efficiency of the ChIP experiments was confirmed by quantitative PCR. Prior to library preparation, 5-10ng ChIP material was diluted to 50ul in TLE buffer (10mM Tris-HCl pH8.0, 0.1mM EDTA) and sonicated with Bioruptor Pico sonicator for 17 min (30 sec on/off). The antibodies used for ChIPseq experiments were rabbit polyclonal anti-CDK8 (A302-500A, Bethyl laboratories, 2.5μl), rabbit monoclonal anti-RING1B (5694, Cell signaling, 3 μl), rabbit polyclonal anti-PCGF2 (sc-10744, Santa Cruz, 3ul), rabbit monoclonal anti-T7-Tag (D9E1X) (13246, Cell signaling, 3ul). The antibodies used for ChIP-qPCR for TetO targeting experiments were rabbit polyclonal anti-FS2 (produced in house, 59, 3ul), polyclonal anti-MED12 (A300-774A, Bethyl laboratories, 3ul), polyclonal anti-MED1 (A300-793A, Bethyl laboratories, 3ul), polyclonal anti-CCNC (A301-989A, Bethyl laboratories, 3ul). For native ChIP, 50x10^6 ESCs were mixed with 20x10^6 SG4 Drosophila cells and washed with 1x PBS prior to chromatin isolation. Nuclei were released in ice cold lysis buffer (10mM Tris-HCl pH 8.0, 10mM NaCl, 3mM MgCl2, 0.1% NP40), washed and resuspended in 1ml ice cold digestion buffer (10mM Tris-HCl pH 8.0, 10mM NaCl, 3mM MgCl2, 0.1% NP40, 0.25M sucrose, 3mM CaCl2, 1x cOmplete protease inhibitor cocktail (Roche)). Chromatin was digested with 200mM MNase (ThermoFisher scientific) for 5 min at 37°C and the reaction was stopped by the addition of 4mM EDTA pH 8.0. The samples were centrifuged at 1500g for 5 min at 4°C, the supernatant (S1) was retained. The remaining pellet was incubated with 300μl of nucleosome release buffer (10mM Tris-HCl pH 7.5, 10mM NaCl, 0.2mM EDTA, 1x protease inhibitor cocktail (Roche)) at 4°C for 1 h, passed five times through a 27G needle using a 1mL syringe, and spun at 1500g for 5 min at 4°C. The second supernatant (S2) was collected and combined with corresponding S1 sample from above. Digestion to mostly mononucleosomes was confirmed on a 1.5% agarose gel. The prepared native chromatin was aliquot, snap frozen in liquid nitrogen and stored at -80°C until further use. ChIPs were performed as described in Fursova et al 2019, using 5ul of H3K27me3 antibody prepared in-house. 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.