characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2015-06-30T17:01:03Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2018-03-08T16:50:28Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA2234965
characteristics.characteristic: External Id
characteristics.text: National Research Center for Epigenome Reprogramming Network
characteristics.characteristic: INSDC center name
characteristics.text: 2015-06-30T17:01:03Z
characteristics.characteristic: INSDC first public
characteristics.text: 2018-03-08T16:50:28Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS364247
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-2002:cell_culture_1
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: mouse embryonic stem cell
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0004038
characteristics.characteristic: cell type
characteristics.text: house mouse
characteristics.characteristic: common name
characteristics.text: Protocols: We purchased normal mouse embryonic stem cells (E14) from the Mutant Mouse Regional Resource Centers (MMRRC). We cultured stem cells in DMEM containing 15% (v/v) FBS (Hyclone), 2 mM L-glutamine, 1% (v/v) nonessential amino acids, 55uM-mercaptoethanol, antibiotics (all from Gibco) and 1,000 U ml-1of ESGRO (Chemicon) on gelatin-coated plates. Cells were cross-linked with 1% (v/v) formaldehyde for 10min at 37C and lysed with buffer containing 85mM KCl, 5mM PIPES (pH 8.0) and 0.5% (v/v) NP-40. After centrifugation, nuclear pellets were resuspended in buffer containing 10mM EDTA, 100mM Tris-Cl (pH 8.1) and 1% SDS. To solubilize and shear cross-linked DNA, lysates were sonicated. 10-fold diluted lysates in IP buffer containing 16.7mM Tris-Cl (pH 8.1), 167mM NaCl, 0.01% (w/v) SDS, 1.1% (v/v) Triton X-100 and 1.2mM EDTA were incubated overnight at 4C with protein A/G PLUS agarose and 1-2ug appropriate antibodies.(Ctbp2 antibody=BD Transduction Laboratories, cat#612044; normal mouse igG antibody = Santa Cruz, cat#sc-2025) Beads were washed with 1X 20mM Tris-Cl (pH 8.1), 150mM NaCl, 0.1% (w/v) SDS, 1% Triton X-100, 2mM EDTA, 1X 20mM Tris-Cl (pH 8.1), 150mM NaCl, 0.1% (w/v) SDS, 1% Triton X-100, 2mM EDTA, 1X 0.25M LiCl, 1% (v/v) NP40, 1% (v/v) Deoxycholate, 1mM EDTA, 10mM Tris-Cl (pH 8.1) and 2X with TE. Immune complexes were then eluted by adding 250ul of elution buffer containing 1% (w/v) SDS and 0.1M NaHCO3 two times. Cross-linking was reversed by adding of 20ul of 5M NaCl and incubated overnight at 65C. DNA was precipitated with ethanol and kept in -20C for the further use.
characteristics.characteristic: description
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: fresh specimen
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/OBI_0000971
characteristics.characteristic: specimen with known storage state
characteristics.text: cell_culture_1
characteristics.characteristic: title