Mus musculus
Protocols: ESCs were cultured as described in GMEM/10% FCS with 100 units/ml LIF on gelatinised tissue culture flasks at a density of 2.5-10x104/cm2. DNA was extracted by phenol/chloroform/isoamyl-alcohol followed by chloroform, then precipitated with ethanol and resuspended in TE buffer. ESCs were fixed with 1% formaldehyde (10 min, rt) and crosslinking stopped by addition of glycine to 0.2 M. Cells were lysed by incubating in 10 mM Tris-HCl, 0.25% Triton X-100, 10 mM EDTA, 100 mM NaCl (twice, 15min, 4 C). Nuclei were lysed in 50 mM HEPES-KOH, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% deoxycholate, 1% SDS (15 min, 4 C), centrifuged and the chromatin pellet resuspended in 0.1% SDS buffer for shearing on ice to an average size of about 250 bp using a probe sonicator (Branson digital sonifier S-450D). Chromatin extracts were pre-cleared with Protein G Dynal Magnetic Beads (Invitrogen) (2 h, 4 C) and immunoprecipitated overnight at 4 C using Protein G Dynal Magnetic Beads pre-coupled with antibodies against Oct4 (Santa Cruz N19, sc8628), Nanog (Cosmo Bio RCAB0002P-F) or H3K27me3 (Millipore 07-449). Beads were washed 3x with 0.1% SDS buffer, once with 0.1% SDS/ 0.35M NaCl buffer, once in 10 mM Tris-HCl, 0.25M LiCl, 1 mM EDTA, 0.5% deoxycholate, 0.5% NP-40, and once in TE buffer (10 mM Tris-HCl, 1 mM EDTA). Immunoprecipitated material was eluted from the beads and crosslinks reversed by incubation with pronase for 2 h at 42 C then 6 h at 67 C. DNA was extracted by phenol/chloroform/isoamyl-alcohol followed by chloroform, then precipitated with ethanol and resuspended in TE buffer. ChIP-Seq library was prepared using the ChIP-Seq Sample Preparation Kit (Illumina) and NEBNext?? ChIP-Seq Library kit (NEB Biolabs) according to the manufacturer's instructions and sequenced for 36 cycles with the HiSeq 2000 system (Illumina).