Instrument: Illumina HiSeq 1500
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: SINGLE
Construction protocol: ChIP-seq was performed on MNase digested chromatin as described previously (Straub et al., 2008 and Kasinathan et al., 2013) with some modifications. Cells were fixed in 1% formaldehyde for 1 min at 26°C, followed by quenching with 125 mM glycine and washing with PBS. Nuclei were released by resuspending in TM2+ with NP-40. MNase digestion was performed in TM2+IC using 4U MNase (Sigma Aldrich, resuspended in EX50 (Bonte & Becker, 1999)) in the presence of CaCl2 for 13 min at 37 °C. Reaction was stopped with EGTA and Triton-X-100, SDS, NaDOC and NaCl was added to final concentration as in RIPA Buffer. MNase digested chromatin was incubated for 1 h at 4°C while slight agitation and chromatin was solubilized by passing ten times through 27 G needle and centrifuged for 30 min at 15,000 g at 4°C. 100 µL soluble chromatin was pre‑cleared with 3 µL (6 µL 50% slurry) Protein A:Protein G (1:1) beads (GE Healthcare) and supernatant was directly used for immunoprecipitation by adding antibody to 200 µL chromatin adjusted to 500 µL with RIPA and incubated for 16 h at 4°C. 100 µL supernatant was added to 3 µL (6 µL 50% slurry) Protein A:Protein G beads (1:1) and icubated for 4 h at 4°C. Beads were washed five times with RIPA. For DNA recovery, beads were resuspended in 6.7 bed volumes TE Buffer (10 mM Tris/HCl pH 8.0, 1 mM EDTA), RNA was digested with 50 µg/mL RNaseA (Sigma‑Aldrich) for 30 min at 37°C, and protein was digested with 0.5 µg/mL Proteinase K (Sigma‑Aldrich) supplemented with 0.5% (v/v) SDS for 16 h at 65°C. DNA was purified with 1.8x AMPure XP beads (Beckmann Coulter). Libraries were prepared with NEBNext ChIP‑Seq Library Perp Kit for Illumina (NEB).