Instrument: Illumina HiSeq 2500
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: SINGLE
Construction protocol: ChIP was performed as previously described (Blecher-Gonen, R. et al. Nat. Protoc. 8, 539-554. 2013). Briefly, 20-50 x 106 cells were fixed in 1% formaldehyde (Sigma-Aldrich) for exactly 10 min at RT under rotation. Crosslinking reaction was quenched by the addition of 125 mM Tris-HCl pH 7.6 for 5 min at RT. Cells were washed 3 times with ice-cold PBS. The cell pellet was then resuspended in RIPA buffer (without Triton X-100) supplemented with protease inhibitors (Halt™ Protease Inhibitor Cocktail, Thermo Scientific) and frozen at -80°C. Chromatin was sheared with a Bioruptor® Plus bath sonicator (Diagenode) applying the following settings: 5 x 10 cycles 30 sec ON and 60 sec OFF, high intensity, at 4°C. Every 10 cycles samples were mixed and briefly spun down. The shearing of DNA into 200-300 bp fragments was evaluated by electrophoresis on a 1.5% agarose gel. Prior to IP, 1% Triton X-100 was added to the sample and an aliquot was stored at -20°C as input control. Antibody-bound Protein G dynabeads™ (Life Technologies) were added to each sample (10 μg c-MAF antibody per sample) and immunoprecipitation was performed overnight at 4°C on a rotating wheel. The day after, dynabeads were thoroughly washed: 6 times with RIPA buffer (10 mM Tris-HCl pH 8.0, 140 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100, 0.1% 1% Na-Deoxycholate), then twice with RIPA-500 buffer (10 mM Tris-HCl pH 8.0, 500 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100, 0.1% 1% Na-Deoxycholate), twice with LiCl wash buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 1 mM EDTA, 0.5% NP-40, 0.5% Na-Deoxycholate), and once with TE buffer. ChIP DNA was eluted with direct elution buffer (10 mM Tris-HCl pH 8.0, 300 mM NaCl, 5 mM EDTA, 0.5% SDS) and subjected to RNAse A and proteinase K treatment before overnight de-crosslinking at 65°C. Finally, de-crosslinked DNA was purified with SPRI magnetic beads and eluted in 10 mM Tris-Cl pH 8.0. ChIP DNA was quantified by picogreen on a Qubit 2.0 device. ChIP-seq libraries were prepared as previously described (Blecher-Gonen, R. et al. Nat. Protoc. 8, 539-554. 2013).