Mus musculus
Protocols : Growth Protocol: For 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. 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 homogenise. 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 sonication 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. Each sample input was incubated with 70 Î¼L of Invitrogen M-280 anti-rabbit beads (Invitrogen) pre-coupled for 6h at 4C with the specific antibody. Samples were incubated overnight at 4C and the following day beads were washed four times with LB3-100 buffer followed by one wash with LB3-500 buffer, followed by four washes with RIPA-250 buffer, finally beads were quickly rinsed with 10 mM Tris/1 mM EDTA buffer.