Anti-Smad3 antibody - ChIP Grade (ab28379)- Lot #- GR155884-8
BJ fibroblasts 5 days post induced viral expression of OSKM
Sequenced DNA Library
10 million cells were cross-linked with 10 mM DTBP (Sigma Aldrich) and 1% formaldehyde for 10 min at room temperature, with the formaldehyde then quenched by the addition of glycine (125mM final concentration) and the cells were then washed twice with ice-cold PBS. Cell lysis was carried out with a buffer containing 10 mM Tris-Cl (pH 8), 100 mM NaCl, 10 mM EDTA, 0.25% Triton X-100 and protease inhibitor cocktail (Roche). Cells were then resuspended in 1% SDS lysis buffer containing 50 mM HEPES-KOH (pH 7.5), 150 mM NaCl, 1% SDS, 2 mM EDTA, 1% Triton X-100, 0.1% NaDOC and protease inhibitor cocktail. The suspension was nutated for 15 mins at 4°C before spinning down to collect the chromatin pellet. The pellet was then washed twice with 0.1% SDS lysis buffer containing 50 mM HEPES-KOH (pH 7.5), 150 mM NaCl, 0.1% SDS, 2 mM EDTA, 1% Triton X-100, 0.1% NaDOC, 1 mM PMSF and protease inhibitor cocktail. Sonication was conducted using the Bioruptor (20 cycles, 30s pulses on with 90s pulses off). The chromatin solution was clarified by centrifugation at 20,000 g at 4°C for 45 mins and then pre-cleared with Dynabeads protein G (Life Technologies) for 2 hrs. The pre-cleared chromatin sample was incubated with 50 μl of Dynabeads protein A loaded with 10 μg antibody overnight. The beads were then washed three times with 0.1% SDS lysis buffer, once with 0.1% SDS lysis buffer/0.35 M NaCl, once with 10 mM Tris-Cl (pH 8), 1 mM EDTA, 0.5% NP40, 0.25 LiCl, 0.5% NaDOC, and once with TE buffer (PH8.0). The immunoprecipitated material was eluted from the beads by heating for 45 min at 68°C in 50 mM Tris-Cl (pH 7.5), 10 mM EDTA, 1% SDS. To reverse the crosslinks, samples were incubated with 1.5 μg/ml Pronase at 42°C for 2 hr followed by 67°C for 6 hr. The samples were then extracted with miniElute DNA purification kit (Qiagen).