characteristics.text: 2011-09-16T17:00:32Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2018-03-08T15:29:02Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA1094169
characteristics.characteristic: External Id
characteristics.text: CEA/Centre National de Genotypage
characteristics.characteristic: INSDC center name
characteristics.text: 2011-09-16T17:00:32Z
characteristics.characteristic: INSDC first public
characteristics.text: 2018-03-08T15:29:02Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS054241
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-767:46C
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: 46C
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0005484
characteristics.characteristic: cell line
characteristics.text: embryonic stem cell
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/CL_0002322
characteristics.characteristic: cell type
characteristics.text: house mouse
characteristics.characteristic: common name
characteristics.text: Protocols: 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 (0.4% 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), pelleted by centrifugation at 2500 rpm and resuspended in 1 ml per dish of FA/SDS buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X100, 0.1% Na-deoxycholate, 0.1% SDS) with protease inhibitors and incubated on ice for 15 min. The subsequent operations were performed at 4C. Chromatin was collected by centrifugation for 20 min at 12 000 rpm, resuspended in the same amount of FA/SDS and put on a rotating wheel for 1 hr, centrifuged and resuspended in 0.4 volume of FA/SDS with protease inhibitors. 400 ul batches of chromatin were fragmented with a sonicator (Diagenode) to generate DNA fragments of 300-400 bp mean size. The chromatin was collected by centrifugation at 14 000 rpm for 10 min and was kept at -80C until needed. The immunoglobulin-coupled magnetic beads (Dynal) used in the chromatin immunoprecipitation experiments were prepared essentially as described previously (Esnault, C., Ghavi-Helm, Y., Brun, S., Soutourina, J., Van Berkum, N., Boschiero, C., Holstege, F. and Werner, M. (2008) Mol. Cell). Anti-HA antibody was HA7 H-3663 (Sigma) and anti-Flag anti-body was F-1804 (Sigma). For each ChIP experiment, 240 ug DNA (RPC1, RPC4, 46C) or 750 ug DNA (BRF1, BRF2) of sonicated chromatin were incubated with 150 ul beads for 2 hr at room temperature. The beads were magnetically pelleted and the supernatant was removed. The beads were washed with 1 ml of low salt buffer (pH 8.0, 20 mM Tris-HCl, 2 mM EDTA, 150 mM NaCl, 1% Triton X-100, 0.1% SDS), then with the same volume of high salt buffer (pH 8.0, 20 mM Tris-HCl, 2 mM EDTA, 500 mM NaCl, 1% Triton X-100, 0.1% SDS), then with low LiCl buffer (pH 8.0, 10 mM Tris-HCl, 1 mM EDTA, 250 mM LiCl, 1% NP-40, 1% deoxycholate) and finally twice with TE (pH 8.0, 10 mM Tris-HCl, 1 mM EDTA). The first ChIP was performed using the anti-HA antibody. The bound chromatin was eluted using FA/SDS buffer containing the HA peptide (0.5 mg/ml; Ansynth Service) for 4 h at 16C then overnight at 4C. For the second ChIP, the supernatant was incubated with 50 ul beads coupled to anti-Flag antibodies which were treated as above except for the elution that was performed by incubation with a buffer containing 1% SDS and 0.1 M NaHCO3. Reversal of the cross-links and DNA purification were performed as described previously (Kuras, L., Borggrefe, T. and Kornberg, R.D. (2003) Proc. Natl. Acad. Sci. USA).
characteristics.characteristic: description
characteristics.text: transfection
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0000726
characteristics.characteristic: geneticmodification
characteristics.text: untagged cell
characteristics.characteristic: genotype
characteristics.text: Mus musculus
characteristics.characteristic: organism
characteristics.text: Mus musculus
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_10090
characteristics.characteristic: organism
characteristics.text: Mus musculus
characteristics.characteristic: scientific_name
characteristics.text: male
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/PATO_0000384
characteristics.characteristic: sex
characteristics.text: 46C
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0005484
characteristics.characteristic: title