Genomic binding of Pol III transcription machinery and relationship with TFIIS distribution in mouse embryonic stem cells
Genomic binding of Pol III transcription machinery and relationship with TFIIS distribution in mouse embryonic stem cells
ES cells were cultured on embryonic fibroblast feeder cells blocked with mitomycin C in D15 medium (Dulbecco Modified Eagle Medium High Glucose supplemented with 15% FBS, 2mML-glutamine, 50 units/ml penicillin, 50 ug/ml streptomycin, 0.1 mM nonessential amino acids (all from GIBCO), 0.1 mM 2-mercaptoethanol [Sigma] and 1,000 units/ml LIF). ES cells were maintained at 37C, 5% carbon dioxide, fed with fresh media daily, and transferred to new plates after trypsinization. Typically 150-200 million cells were collected for each ChIP experiment. The cellular proteins and DNA were cross-linked by the addition of formaldehyde (1% final concentration) in cell culture media. The plates were incubated for 10 min at room temperature then, the reaction was stopped by the addition of glycine (0.125 M final concentration). The cells were washed twice with 10 ml chilled PBS. Each plate was scraped with 2 ml PBS with protease inhibitors (Roche complete, 10 mM PMSF dissolved in ethanol). Untagged ES cells (46C-2), TFIIIC subunits and TCEA1 ChIPs were fragmented using MNase I. The protocol will be described in details elsewhere (M. Gerard, manuscript in preparation).
Experimental Factor: IMMUNOPRECIPITATE
Chromatin IP in untagged 46C
Experimental Factor: GENOTYPE
untagged cell