GSM5320864: Input DNA ESC WT; Mus musculus; ChIP-Seq
For ChIP-seq, approximately 20x106 ESCs were cross-linked by addition of formaldehyde to 1% for 10 min at RT, quenched with 0.125 M glycine for 5 min at RT. Nuclear extracts were prepared as described in Native RIP protocol and were sonicated using the Bioruptor Twin (Diagenode) (20 cycles, 30s on/off) at high-power setting in SDS ChIP Buffer (20 mM Tris-HCl pH 8.0, 10 mM EDTA, 0.5% SDS and protease inhibitors). Cell lysate was diluted with ChIP dilution buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton) before the immunoprecipitation with 2 ug of Histone H3, acetyl-K27 antibody (Abcam) or 2 ug of p300 antibody (Millipore) overnight at 4°C on a rotator.   Protein-DNA complexes were immobilized on protein G-Dynabeads (Life technologies) and washed 6 times with RIPA buffer (500 mM LiCl, 50 mM HEPES-KOH pH 7.6, 1 mM EDTA, 1% NP-40, 0.7% Na-Deoxycholate). The de-crosslinking was performed at 65°C overnight. De-crosslinked DNA was purified using QiaQuick PCR Purification Kit (Qiagen), according to the manufacturer's instruction. To prepare ChIP-seq libraries, approximately 10 ng of purified ChIP DNA from two replicates of WT and two replicates of lncSmad7 KO ESCs were end-repaired, dA-tailed, and adaptor-ligated using the NEBNext ChIP-seq Library Prep Master Mix Set (NEB), according to the manufacturer's instructions. The data are expressed as a percentage of the DNA Input.