Chromatin immunoprecipitation was performed as described previously (Farcas et al., 2012) with slight modifications. Cells were fixed for 45 min with 2mM DSG (Thermo scientific) in PBS and 12.5 min with 1% formaldehyde (methanol-free, Thermo scientific). Reactions were quenched by the addition of glycine to a final concentration of 125µM. After cell lysis and chromatin extraction, chromatin was sonicated using a BioRuptor Pico sonicator (Diagenode), followed by centrifugation at 16,000×g for 20min at 4°C. 200µg chromatin diluted in ChIP dilution buffer (1% Triton-X100, 1mM EDTA, 20mM Tris-HCl (pH 8.0), 150mM NaCl) was used per IP. 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 previously (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 20ug/ml Proteinase K and purified using ChIP DNA Clean&Concentrator™ kit (Zymo research). ChIP DNA was sheared with Bioruptor sonicator to generate DNA fragments between 100-350bp. Libraries were prepared using the NEBNext Fast DNA fragmentation and library preparation kit for Ion Torrent (NEB, E6285S) following manufacturer's instructions. Briefly, 30-40ng ChIP or input DNA material was used. Libraries were size-selected for 250bp fragments using 2% E-gel SizeSelect gel (Thermo scientific) . Templates were generated with Ion PI™ Template OT2 200 kit v3 and Ion PI™ Sequencing 200 kit v3, or with Ion PI IC 200 kit (Thermo scientrific). Libraries were sequenced on the Ion Proton Sequencer using Ion PI™ chips v2 (Thermo scientific)
GEO Accession
GSM2611270