Instrument: Illumina HiSeq 2000
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: PAIRED
Construction protocol: Cells were lysed by resuspending the cells in lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, protease inhibitors) and bead beating with 0.7 mm zirconium beads for six rounds of 2 minutes, interspersed with cooling on ice. Cells were collected by centrifugation, the supernatant was discarded, and the pellet resuspended in MNase Digestion buffer (50 mM Tris pH 7.5, 100 mM NaCl, 5 mM MgCl2, 1 mM CaCl2, 0.1% Triton X-100). Chromatin was digested with MNase at 37C for 15 minutes. The amount of MNase was empirically determined to yield an estimated 60% mono-, 30% di-, and 10% tri-nucleosomes. MNase digestion was stopped by the addition of 0.1X volumes of 10X MNase ChIP Stop Buffer (50 mM Tris pH 7.5, 500 mM NaCl, 100 mM EDTA, 20 mM EGTA, 10% Triton X-100, 1% sodium deoxycholate). Cell debris was removed by centrifugation and the soluble chromatin was added to prepared beads. For immunoprecipitation, anti-mouse magnetic Dynabeads (Invitrogen) were prepared by incubating with anti-cMyc (9E11) in phosphate-buffered saline containing 5 mg/ml BSA for several hours with rotation. Normalized amounts of protein was then incubated with the prepared beads in ChIP buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate). Immunoprecipitations were performed at 4C overnight with gentle rotation. Beads were harvested by magnetic field and washed twice with ChIP buffer, twice with ChIP buffer supplemented to 500 mM NaCl, twice with LiCl wash buffer (10 mM Tris pH 8, 250 mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA), and once with TE. Captured complexes were eluted and cross-links reversed by incubating the beads in buffer (10 mM Tris pH 8, 1 mM EDTA, 0.5% SDS) at 65C overnight. DNA was purified using Qiagen PCR Clean-up columns using the manufacturer's protocol. ChIP eluate DNA and Input DNA were phosphorylated with T4 kinase (NEB) prior to ligation of adaptors using Illumina Tru-Seq library construction kit according to manufacturers instructions. No other end repair was performed (fill-in, etc). After limited PCR amplification, samples were size selected on agarose gel and fragments corresponding to mononucleosomes were excised and purified.