BioSample

Protocols : Growth Protocol: For Setd5, PolII, and Hdac3 each replicate 100 million ESCs were harvested and cross linked in 1.5 mM EGS in phosphate-buffered saline (PBS) for 1h followed by 10 min cross linking in 1.5% paraformaldehyde (PFA) in PBS at room temperature. For H3K27ac and H4pan acetyl 20million cells were harvested and crosslinked in 1% PFA for 10 minutes. Cross-linking solution was quenched with 125 mM Glycine for 5 min. Fixed cells were spun down and washed twice with ice cold PBS freshly supplemented with protease inhibitors.Samples were snap frozen in liquid nitrogen and stored at -80C. Chromatin was prepared by re-suspending cross linked cells in hypotonic buffer on ice, followed douncing with a tissue homogenize. Samples were spun down and nuclear pellets were re-suspended in MNase digestion buffer. MNase digestion was performed at for 5 minutes 37C. MNase enzymatic activity was quenched by adding EDTA/EGTA based quenching buffer. Digested chromatin was sonicated with 3 cycles of sonicator at 4C.Samples were spun down to separate insoluble chromatin. Soluble fragmented chromatin was used as ChIP input. Both soluble input and precipitated pellet were resolved on an agarose DNA gel to check the digestion pattern across replicates and samples. 5% of each chromatin inputs were set aside before the IP.