NuRD-mediated gene repression in mouse embryonic stem cells
NuRD-mediated gene repression in mouse embryonic stem cells
ES cells were grown in GMEM supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate, 2 mM L-glutamine, 0.1 mM non-essential amino acids and 10 ng/ml murine LIF. Eed-/- ES cells were cultured on mitotically inactivated primary mouse embryonic fibroblasts which were depleted from ES cells upon harvest. TSA (Sigma) was added to normal ES cell media to a final concentration of 0.1 mM for 2 h prior to harvesting. Cells were fixed using 1% formaldehyde for 10min at room temperature and fixation quenched with 150 mM glycine. For Mi2beta ChIP-seq, formaldehyde fixation was preceded by 45 min incubation in 2 mM disuccinimidyl glutarate (Sigma). Chromatin was sheared by sonication to an average fragment size between 200 and 300 bp using a Bioruptor sonication instrument (Diagenode).
Experimental Factor: GENOTYPE
Mdb3 -/-
Experimental Factor: IMMUNOPRECIPITATE
H3K27me3