characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2017-10-31T17:01:50Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2017-05-16T18:32:04Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA104041898
characteristics.characteristic: External Id
characteristics.text: unspecified
characteristics.characteristic: INSDC center name
characteristics.text: 2017-10-31T17:01:50Z
characteristics.characteristic: INSDC first public
characteristics.text: 2017-05-16T18:32:04Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS1705791
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-5732:CTCF_ES_2i_rep1_CTCF
characteristics.characteristic: Submitter Id
characteristics.text: Millipore 07-729
characteristics.characteristic: antibody
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: 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: ES cells were grown at 37 °C in a 5% (v/v) CO2 on 0.1% (v/v) gelatin-coated flasks in the presence of GSK3ß and MEK inhibitors and LIF (2i/LIF medium). Complete medium comprised N2B27 (50% Dulbecco’s Modified Eagle’s Medium (DMEM), 50% F12 (DMEM/F12, Invitrogen), 2.5 ml of N2 and 5ml of B27 (Gibco), BSA (Gibco; final concentration 0.012%), non-essential amino acids (Gibco), glucose (final concentration 0.03 M), HEPES (final concentration 4.5 mM) and 0.1 mM beta-mercaptoethanol) was supplemented with CHIR99021, an inhibitor of GSK3ß, at a final concentration of 3µM (Trevigen) and PD0325901, a MEK inhibitor, at the final concentration of 1 µM (Trevigen) and 2 ng/ml LIF. As noted below, differentiation of ES cells from the naive state can be obtained by simple withdrawal of the 2i and LIF factors. Cells were crosslinked with 1% formaldehyde for 10min at RT, glycine was added to a final concentration of 0.125M to quench formaldehyde. Cells were lysed with 1 ml of lysis buffer (10 mM Tris pH 7.5, 1 mM EDTA, 0.1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100, 1× Complete Mini EDTA free proteinase inhibitors) for 20 minutes on ice. Chromatin was sonicated to average fragment size of 400bp. Chromatin extracts corresponding to 10M of cells (90µg of DNA) were precleared with 40µl of Protein A dynabeads. The sample was incubated at 4°C with overhead mixing for 1 hour. 40µl of protein A Dynabeads was washed with PBS and incubated with anti-CTCF (Milipore, 07-729, 5µl) or anti-Rad21 (Abcam, ab-992, 5µg) at RT with overhead mixing (40µl of beads per 5µl of anti-CTCF or 5µg of anti Rad21). 500µl of pre-cleared chromatin was mixed with the antibody-coupled beads and incubated overnight at 4°C with overhead mixing. Next, beads were washed for 10 minutes at 4°C with overhead mixing with lysis buffer (2x), lysis buffer containing 0.3M NaCl (2x), LiCl buffer (0.25 M LiCl, 0.5% Igepal-630, 0.5% sodium deoxycholate, 2x), TE (pH 8.0) plus 0.2% Triton X-100, and TE (pH 8.0). Beads were resuspended in 100µl of TE (pH 8) and incubated at 65°C for 14 hours, 1µl of RNAseA was added and the sample incubated for 1 hour at 37°C. Next,10µl of Proteinase K was added and the sample incubated at 55°C for 2 hours. Beads were separated from the solution using a magnet. 110µl of the supernatant was mixed with 200µl of Ampure Beads (Beckman Coulter) and DNA was extracted following manufacturer’s suggestions. The Library was prepared in Ovation SP Ultralow library system (Nugen) using 2ng of ChIP material.
characteristics.characteristic: description
characteristics.text: 2i_LIF
characteristics.characteristic: growth condition
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: naive embryonic stem cell
characteristics.characteristic: progenitor cell type
characteristics.text: Biological_replicate1
characteristics.characteristic: replicate
characteristics.text: Mus musculus
characteristics.characteristic: scientific_name
characteristics.text: CTCF_ES_2i_rep1_CTCF
characteristics.characteristic: title