characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2013-12-05T17:02:14Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2018-03-08T16:51:00Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA2234722
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: 2018-03-08T16:51:00Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS363974
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-2031: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, passage 28) 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. hESCs cultured in TeSR1 were treated with 3iL 48hrs 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 is refreshed daily and cells are subcultured upon confluency. For expression analysis, total RNA from the cells was extracted using the TRIzol reagent (Invitrogen). Total RNA was extracted with chloroform and precipitated with isopropanol. The precipitated RNA was centrifuged at 13000rpm at 4 degrees C for 10mins. The RNA pellet was washed with 70% ethanol and subsequently reconstituted with DEPC-treated water (AMBION). DNA contaminants were digested with DNASE I (Ambion) at 37 degrees C for 30 min. The total RNA was further purified by column (Purelink RNA Mini kit, Ambion). RNA-Seq libraries were prepared using 4 ug of total RNA according to the manufacturer (TruSeq RNA Sample Preparation Kit v2, Illumina). Briefly, mRNA was obtained using two rounds of purification with poly-T oligo-attached magnetic beads. The mRNA was then fragmented and subjected to the first strand and second strand cDNA synthesis, then end-repaired, adenylated, adaptor ligated and finally subjected to 15 cycles of PCR.
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