Chromatin immunoprecipitation (ChIP)

Crosslinking was performed at room temperature with 1% formaldehyde (Sigma) for 5 min and quenched with 125 mM glycine (ICN Biomedical) for 10 min. Cell membranes were lysed using cold NP-40 buffer (1% NP40, 150 mM NaCl, 50 mM Tris–HCl; pH 8.0) and nuclei collected by centrifugation at 12 000 × g for 1 min at 4 °C. Nuclear pellets were resuspended in ChIP sonication buffer (1% SDS, 10 mM EDTA, 50 mM Tris–HCl; pH 8.0), supplemented with Halt protease inhibitors (Thermo Scientific), and chromatin sheared to an average size between 150 and 500 bp by sonication (Bioruptor Twin, Diagenode). Chromatin preparations were cleared by centrifugation at 20 000 × g for 10 min at 4 °C and protein content measured by BCA assay (Thermo Scientific). 250 μg (H3K36me3 ChIP) or 100 μg (H3K9me3 ChIP) of chromatin was diluted at least 10-fold in ChIP dilution buffer (1.1% Triton X-100, 0.01% SDS, 167 mM NaCl, 1.2 mM EDTA, 16.7 mM Tris–HCl; pH 8.1), the appropriate antibody was added, and samples were incubated overnight at 4°C with rotation. Immune complexes were captured using protein G (Millipore, #16-201) or protein A/G (SantaCruz Biotechnology, #sc-2003) agarose beads blocked with BSA and salmon sperm DNA, and washed with ChIP dilution buffer. Washed beads were resuspended in crosslink reversal buffer (50 mM EDTA, 10 mM Tris–HCl; pH 8.0) and incubated at 95°C for 10 min. DNA was purified by column purification (QIAGEN) and qPCR was performed using SYBR Green chemistry (Quanta or Roche) and gene-specific primers. For ChIP-analysis in 293 T-REx host cells, H3K36me3 and H3K9me3 enrichment were determined relative to pan-histone H3 [2^{(CT_pan-H3 – CT_H3K36me3 or CT_H3K9me3)}] and normalized to a control amplicon in the vector-introduced and non-expressed Amp gene to correct for variations in IP efficiency. Pan-histone H3 occupancy was determined relative to input [% input = 100 × 2^{(CTinput-CTIP)}] and normalized to H3 levels at a control locus in the Amp gene. In the 293T cellular background, H3K36me3 and H3K9me3 occupancies were calculated relative to input using the above formula. Each ChIP-qPCR experiment was performed on three biological replicates and the data were plotted as mean ± SEM, unless otherwise noted. The following antibodies were used for ChIP: histone H3 (Cell Signalling Technology, #2650; 4 μl per IP), H3K36me3 (abcam, #ab9050; 2 μg per IP), H3K9me3 (abcam, #ab8898; 2 μg per IP), normal rabbit IgG (Cell Signaling Technology, #2727; 2 μl per IP). Primers used for ChIP-qPCR analysis are presented in Supplementary Table S1.