RNA sequencing (RNA-Seq) and chromatin immunoprecipitation followed by sequencing (ChIP-seq)

RNA was extracted with Trizol (Invitrogen) and subsequently cleaned and DNase treated using RNeasy columns (Qiagen). DNase-treated RNA was subjected to library preparation. Libraries for RNA-Seq were prepared with ScriptSeq complete Gold kit (Epicenter) and subjected to a 75 bp paired-end sequencing run on NextSeq 500, using Illumina’s NextSeq 500 high output sequencing kit following the manufacturer’s instructions.

For ChIP-seq, cells were cross-linked with 1% formaldehyde for 10 min, followed by quenching with 125 mM glycine for 5 min. Fixed cells were resuspended in cell lysis buffer (10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 0.5% NP-40) and incubated on ice for 10 min. The lysates were washed with MNase digestion buffer (20 mM Tris-HCl, pH 7.5, 15 mM NaCl, 60 mM KCl, 1 mM CaCl₂) once and incubated for 20 min at 37 °C in the presence of 1000 Gel units of MNase (NEB, M0247S) in 250 μL reaction volume. After adding the same volume of sonication buffer (100 mM Tris-HCl, pH 8.1, 20 mM EDTA, 200 mM NaCl, 2% Triton X-100, 0.2% sodium deoxycholate), the lysates were sonicated for 5 min (30 sec-on/30 sec-off) in a Diagenode bioruptor and centrifuged at 15,000 rpm for 10 min. The cleared supernatant equivalent to 2–4 × 10⁶ cells was incubated with 2.5 μg of anti-EZH2 antibody (Cell Signaling, Cat. No. 5246) or 2 μg anti-H3K27Me3 antibody (Cell Signaling, Cat. No. 9733) on a rocker overnight. Bound chromatin was eluted and reverse cross-linked at 65 °C overnight. For next-generation sequencing, ChIP-seq libraries were prepared from 10 ng of ChIP and input DNA with the Ovation Ultralow DR Multiplex system (NuGEN). The ChIP-seq libraries were sequenced in a 51 bp paired-end run using the Illumina HiSeq 2000.