ChIPs were performed on formaldehyde cross-linked chromatin isolated from cells grown on 10 cm dishes to ∼80% confluency. Briefly, formaldehyde was added to the attached cells suspended in Phosphate Buffered Saline (PBS) at a final concentration of 1% and the cells were incubated at room temperature for 10 min with shaking. The reaction was stopped by addition of glycine to a final concentration of 0.125 M. Approximately 2 × 107 cells were washed twice in ice-cold PBS, centrifuged and resuspended in lysis buffer 1 (50 mM HEPES pH 8, 10 mM NaCl, 1 mM EDTA, 10% Glycerol, 0.5% NP-40 and 0.25% Triton X-100) for 90 min at 4 °C. Cells were lysed in lysis buffer 2 (10 mM Tris–HCl pH 8.0, 200 mM NaCl, 1 mM EDTA and 0.5 mM EGTA) for 60 min at 4 °C. The chromatin was sheared in sonication buffer (10 mM Tris–HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% sodium deoxycholate and 0.5% N-lauroylsarcosine) to an average size of 200–600 bp using the Bioruptor™. For each immunoprecipitation, 100 μg of sonicated chromatin were diluted in a final volume of 1 ml with dilution buffer and pre-cleared with 30 μl protein A/G agarose beads (SantaCruz) for 4 h at 4 °C on a rotating wheel. Anti-SMAD1 antibody or rabbit IgG were added to the pre-cleared chromatin and incubated overnight at 4 °C on a rotating wheel. Chromatin was precipitated with 30 μl protein A/G agarose beads for 4 h at 4 °C with rotation. The beads were then washed five times with 500 μl RIPA buffer (10 mM Tris–HCl pH 8.0, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% sodium deoxycholate, 1% Triton X-100 and 0.1% SDS) and once with each of the following buffers: WASH buffer (50 mM HEPES, 0.5% sodium deoxycholate, 1% Triton X-100, 1 mM EDTA, 500 mM NaCl and 0.2% NaN3), LiCl buffer (0.25 M LiCl, 0.5% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA and 10 mM Tris pH 8) and TE buffer (10 mM Tris pH 8, 1 mM EDTA). The bound chromatin was eluted in 100 μl TE buffer. Crosslinks were reversed by incubation O.N. at 65 °C after addition of 1 μl RNAse cocktail (Ambion) and 2 h at 50 °C after addition of 2.5 μl SDS 20% + 2.5 μl 20 mg/ml proteinase K (Sigma). The DNA was extracted by using the QIAquick PCR Purification Kit (Qiagen). 2 ng DNA of immunoprecipitated and input chromatin were used for Illumina library preparation. Libraries were generated by using the NuGen Ovation Ultralow Library System v2 Kit.