NURF301-ChIP
150 million cells were fixed by addition of 1% formaldehyde for 15 minutes at 25C, pelleted and washed 3X in ice-cold 1XPBS prior to storage at -80C. Cell pellets were resuspended in 150 L ChIP Lysis Buffer (1% SDS, 10 mM EDTA) and sonicated for 12.5 minutes using a Diagenode Bioruptor sonicator (cycles of 30s on/off, hi power). Samples were diluted 10 fold in ChIP dilution buffer. Samples were pre-cleared using Protein G-conjugated Dynabeads (Invitrogen) for 30 minutes at room temperature, followed by incubation with antibody coated Protein G-conjugated Dynabeads (Invitrogen) for 3 hours at 4C. Immune complexes were recovered by magnetic selection, and washed once with low salt buffer (20 mM Tris (pH 8.1), 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X-100), once with High salt buffer (20 mM Tris (pH 8.1), 500 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X-100), once with LiCl immune complex wash buffer (10 mM Tris (pH 8.1), 1 mM EDTA, 0.25 M LiCl, 1% IGEPAL CA-630, 1% deoxycholic acid) and twice with TE buffer for five minutes each at room temperature. ChIP DNA was eluted using two washes of elution buffer (1% SDS, 0.1 M NaHCO3) for 15 minutes at room temperature. Cross-links were reversed as described above and ChIP DNA purified using 1.8 volumes Agencourt AMPure XP beads (Beckman Coulter). DNA was end-repaired and sequencing libraries prepared using a SOLiD Fragment Library Construction Kit (Life Technologies). ChIP DNA was barcoded using the SOLiD Fragment Library Barcoding Kit Module 116. Sequencing libraries were run on a SOLiD 4 genome analyzer.