title: Genomic binding of Pol III transcription machinery and relationship with TFIIS distribution in mouse embryonic stem cells
design: Genomic binding of Pol III transcription machinery and relationship with TFIIS distribution in mouse embryonic stem cells
library_construction_protocol: 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).
attributes.tag: Experimental Factor: IMMUNOPRECIPITATE
attributes.value: Chromatin IP in untagged 46C
attributes.tag: Experimental Factor: GENOTYPE
attributes.value: untagged cell