Ezh2 and H3K27me3 chromatin immunoprecipitation (ChIP)-Seq

For Ezh2 and H3K27me3 ChIP seq, wild-type CD8⁺ T cells (from n = 4 mice) were activated in vitro with plate-bound anti-CD3 and anti-CD28 antibodies for 4 days and sorted by flow cytometry to exclude dead cells. 4 × 10⁶ CD8⁺ T cells were crosslinked in 1% formaldehyde and ChIP performed using the EZ-Magna ChIP kit (Millipore) according to the manufacturer’s instructions. Briefly, nuclear extracts were prepared and chromatin sheared to an average size of 300 bp using a Covaris E220 hydro-shearing instrument. For each immunoprecipitation (IP), chromatin from 1 million cells and 3 μg of antibody were used. Antibodies used were: rabbit anti-Ezh2 antibody (H-80; Santa Cruz Biotechnology), rabbit anti-H3K27me3 (Millipore), mouse anti-RNA polymerase II (Millipore), and mouse and rabbit normal IgG. Sequence-indexed libraries were prepared from immunoprecipitated DNA and input controls (1%) using the NEB Next ChIP Library Preparation Reagent Set (NEB), according to the manufacturer’s instructions. Library amplification by PCR used 10 cycles for pol II IPs, 12 cycles for input controls and H3K27me3 IPs, 14 cycles for Ezh2 IPs, and 17 cycles for IgG controls. For the H3K27me3 coverage comparison between wild-type and Ezh2-deficient cells, chromatin from 500,000 cells was used and amplified for 14 cycles (H3K27me3 IPs) or 17 cycles (IgG controls). Amplification yielded 200–600 fmoles per sample. Two hundred fmoles of each library were pooled, size-selected to 250–650 bp on a PippinPrep instrument (Sage Science), and sequenced to a depth of 30 million reads (50 nt SE) on an Illumina HighSeq4000 instrument.