GSM1810980: Smad1/5 ChIP-seq, mESCs, LIF+BMP-4; Mus musculus; ChIP-Seq
ChIP using anti-Smad1/5 antibody were performed essentially as described  (Koinuma et al, Mol Cell Biol, 29, 172-186, 2009). SMAD1/5-DNA complexes were isolated with antibody. Briefly, mESCs and mESD-EpiSCs were cultured in 100-mm plates and three to four plates (approximately 5x107 cells) were used for one immunoprecipitation. Cells were fixed with 1% formaldehyde for 10 min at RT with swirling. Cross-linking was stopped by addition of glycine to a final concentration of 125 mM and washed twice with ice-cold PBS. The cross-linked cells were harvested by scraping, pelleted, and resuspended in 1 ml of lysis buffer containing 50 mM Tris-HCl (pH 8.0), 10 mM EDTA, 1% SDS, and protease inhibitor cocktail (P8340, Sigma-Aldrich). Chromatin was sonicated using a Bioruptor sonicator (Diagenode) for 5 min in a cycle of 30 sec ON and 30 sec OFF at ‘high’ power, in order to shear DNA with an average fragment size of 200-500 bp. Cell extract was diluted 10-fold in ChIP dilution buffer [20 mM Tris-HCl (pH 8.0), 2 mM EDTA, 1% Triton X-100, 150 mM NaCl, cOmplete EDTA-free protease inhibitor cocktail]. Anti-mouse IgG Dynabeads (Invitrogen Life Technologies) were preincubated with 10 µg of antibody at 4°C overnight. Chromatin was incubated with prepared beads at 4°C for more than 8 hr. The beads were then washed five times with RIPA buffer [50 mM HEPES-KOH (pH 7.0), 0.5 M LiCl, 1 mM EDTA, 0.7% deoxycholate, 1% NP-40] and once with TE buffer (pH 8.0). Immunoprecipitated samples were eluted and reverse cross-linked by incubation in elution buffer [50 mM Tris-HCl (pH 8.0), 10 mM EDTA, 1% SDS] at 65°C overnight. Genomic DNA was then extracted with a QIAquick PCR purification kit (Qiagen). The library was prepared using NEBNext ChIP-Seq Library Prep Reagent Set for Illumina (New England Biolabs).