Instrument: Illumina HiSeq 2000
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: PAIRED
Construction protocol: Cells were fixed with 1% formaldehyde for 8 min, quenched with 1.25 M glycine, washed in PBS. Cells were resuspended in cells lysis buffer (50 mM Tris pH 8.0, 10 mM EDTA, 1% SDS, 1x Roche Complete Protease inhibitor), kept in ice for 10 min, and sonicated using Bioruptor. Following sonication insoluble material was pelleted by centrifugation at 16000g for 10 minutes at 4°C. A small sample of soluble crosslinked chromatin was purified, checked for correct sonication size range. 25 ug of chromatin was diluted to 350ul with RIPA buffer (10 mM Tris pH 7.5, 140 mM NaCl, 1mM EDTA, 0.5 mM EGTA, 1% Triton, 0.1% SDS, 0.1% sodium deoxycholate, 1x Roche Complete Protease inhibitor). 10 μl of Protein A Dynabeads (Invitrogen) prebound with 2 μg of histone marks antibodies were added. Samples were incubated overnight at 4°C with rotation. Beads were precipitated on magnet stand and washed 3 times with 1 mL of RIPA buffer and once with TE. Crosslink was reversed with 250 μl of elution buffer (20 mM Tris pH 7.5, 5 mM EDTA, 50 mM NaCl, 1% SDS, 5 μg/mL proteinase K) at 65°C for 2 hours. DNA was purified with Qiagen PCR cleanup kit. Concentration of eluted chromatin was measured with Qubit dsDNA HS Assay Kit (Life Technology). Sequencing libraries were prepared using the Illumina TruSeq DNA Sample Preparation Kit according to the manufacturer's protocol. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250-450 bp (insert plus adaptor) were band isolated from an agarose gel. DNA fragments were sequenced using paired-end sequencing technology on Illumina HiSeq 2000 platform.