Chromatin Immunoprecipitation (ChIP) Protocol

Chromatin IP (ChIP) was performed as described previously^{16 (link)}. Briefly, crosslinked and isolated nuclei were sonicated using a Diagenode Bioruptor to an average size of ~250 bp. After pre-clearing with BSA-blocked protein G Sepharose, chromatin was incubated with antibodies at 4°C overnight. The chromatin immunocomplexes were recovered with the same BSA-blocked protein G beads. For ChIP-seq library construction, 5–10 ng of DNA extracted from the chromatin immunocomplexes as described previously^{15 (link)}. Libraries were prepared according to manufacturer's instructions (Illumina) and as described^{15 (link)}. Immunoprecipitated DNA was first end-repaired using End-It Repair Kit (Epicenter), tailed with an A using Klenow exo minus (NEB M0212), and ligated to custom adapters with LigaFast (Promega, city, state #M8225). Fragments of 350±50 bp were size-selected and subjected to ligation-mediated PCR amplification (LM-PCR), using Phusion DNA polymerase (NEB M0530). Libraries were quantified with quantitative PCR using primers annealing to the adapter sequence and sequenced at a concentration of 7 pM on an Illumina HiSeq 2000. All sequencing data has been deposited into GEO/NCBI with the accession number GSE60411.