characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2014-06-04T17:02:57Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2018-03-08T17:13:02Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA2385700
characteristics.characteristic: External Id
characteristics.text: GIS
characteristics.characteristic: INSDC center name
characteristics.text: 2014-06-04T17:02:57Z
characteristics.characteristic: INSDC first public
characteristics.text: 2018-03-08T17:13:02Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS412002
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-2365:mESCs 2i
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: E14
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0007075
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: Feeder-free E14 mouse embryonic stem cells were cultured on 0.1% gelatin under LIF/Serum or 2i culture conditions for three passages Feeder-free E14 mouse embryonic stem cells were cultured on 0.1% gelatin under LIF (1000U Leukemia inhibitory factor)/Serum (ES grade FBS) or 2i (1uM PD0325901 and 3uM CHIR99021, termed 2i) culture for three passages Crosslinking of protein and DNA was done at room temperature for 10 minutes using ESC media that contains 1% formaldehyde. The crosslinking reaction was quenched with 0.2M final concentration of glycine for 5 minutes. Cells were washed extensively with TBSE (20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA), scraped and pelleted. The cells were lysed with 0.1% SDS buffer and collected nuclei were lysed with 1% SDS buffer. Pelleted chromatin was resuspended in the 0.1% SDS buffer (50 mM HEPES-KOH, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% deoxycholate, 0.1% SDS) and the DNA was sheared with the probe sonicator (Branson digital sonifier S-450D) for 18 cycles of 30 seconds with 1 min intervals at amplitude 40. The chromatin sample was centrifuged at 20,000 rpm for 45 mins before the supernatant was harvested as chromatin extracts for ChIP. Chromatin extracts were pre-cleared with Protein G Dynal Magnetic Beads (Invitrogen) (2 h, 4C) and immunoprecipitated overnight at 4C using Protein G Dynal Magnetic Beads pre-coupled with antibodies against Klf2 (antibody serum raised in rabbit against amino acids 90-255 of mouse Klf2) (Jiang et al., 2008, Nature Cell Biology). Beads were washed 3x with 0.1% SDS buffer, once with 0.1% SDS/ 0.35M NaCl buffer, once in 10 mM Tris-HCl, 0.25M LiCl, 1 mM EDTA, 0.5% deoxycholate, 0.5% NP-40, and once in TE buffer (10 mM Tris-HCl, 1 mM EDTA). Immunoprecipitated material was eluted from the beads with 50 mM Tris-HCl, 10 mM EDTA, 1% SDS at 69C for 30 mins in a orbital shaker at 1000 rpm. Crosslinks were reversed by incubation with pronase for 2 h at 42C then 6 h at 67C. DNA was extracted by phenol/chloroform/isoamyl-alcohol followed by chloroform, then precipitated with ethanol and resuspended in TE buffer. ChIP-Seq library was prepared using the NEBNext ChIP-Seq Library kit (NEB Biolabs) according to the manufacturer’s instructions. Briefly, the DNA was end-repaired with exonucleases and an adaptor was ligated to the end. Size selection was performed using AMPure XP Beads prior to PCR amplification for 18 cycles. The amplified DNA was purified using AMPure XP Beads, multiplexed and sequenced with the HiSeq 2000 system (Illumina).
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: male
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/PATO_0000384
characteristics.characteristic: sex
characteristics.text: mESCs 2i
characteristics.characteristic: title