characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2013-12-05T17:02:14Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2018-03-08T16:51:15Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA2235071
characteristics.characteristic: External Id
characteristics.text: GIS
characteristics.characteristic: INSDC center name
characteristics.text: 2013-12-05T17:02:14Z
characteristics.characteristic: INSDC first public
characteristics.text: 2018-03-08T16:51:15Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS364387
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-2041:hESCs
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: H1
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0003042
characteristics.characteristic: cell line
characteristics.text: embryonic stem cell
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/CL_0002322
characteristics.characteristic: cell type
characteristics.text: human
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_9606
characteristics.characteristic: common name
characteristics.text: Protocols: The hESC lines H1 (WA-01) was used for this study. For routine culture of hESCs in TeSR1 (Stem cell technologies), cells were cultured feeder free on matri-gel (BD). Medium was changed daily. The hESCs were subcultured with 1 mg/ml Dispase (Stem cell technologies) every 5-7 days. For treatment with 3iL conditions, hESCs cultured in TeSR1 was treated with 3iL 48 hrs post-seeding. The cells are subsequently subcultured on mitomycin C inactivated mouse fibroblast. Cells are dissociated to single cells using TrypLE (Life technologies). 3iL medium was refreshed daily and cells were subcultured upon confluency. Crosslinking of protein and DNA was done at room temperature for 10 minutes using serum free 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 20 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 H3K4me3 (Millipore 04-745), H3K27me3 (Millipore 07-449) or H3K27ac (abcam ab4729). 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 10000 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 15-18 cycles. The amplified DNA was purified using AMPure XP Beads, multiplexed and sequenced with the HiSeq 2000 system (Illumina).
characteristics.characteristic: description
characteristics.text: Homo sapiens
characteristics.characteristic: organism
characteristics.text: Homo sapiens
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_9606
characteristics.characteristic: organism
characteristics.text: Homo sapiens
characteristics.characteristic: scientific_name
characteristics.text: male
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/PATO_0000384
characteristics.characteristic: sex
characteristics.text: hESCs
characteristics.characteristic: title