characteristics.text: 2013-02-08T17:03:00Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2018-03-08T16:09:43Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA1712776
characteristics.characteristic: External Id
characteristics.text: Pediatric Diabetes Research Center, UCSD Stem Cell Program
characteristics.characteristic: INSDC center name
characteristics.text: 2013-02-08T17:03:00Z
characteristics.characteristic: INSDC first public
characteristics.text: 2018-03-08T16:09:43Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS197530
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-1086:DP3
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: Cyt49
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: CyT49 hESCs were maintained and differentiated as previously described [DAmour, K.A., et al., Nat Biotechnol, 2006. 24:1392-401; Kroon, E., et al., Nat Biotechnol, 2008. 26:443-52; Kelly, O.G., et al., Nat Biotechnol. 29:750-6]. Non-adherent conditions were applied to hESC differentiation. Briefly, cells were lysed in Trizol reagent (Invitrogen) for extraction of total RNA. Residual contaminating genomic DNA was removed using the Turbo Dnase kit (Ambion). mRNA was isolated from 2 ug of DNA-free total RNA using the Dynabeads mRNA Purification kit (Invitrogen). Strand-specific RNA-seq libraries were prepared as previously described [Parkhomchuk, D., et al., Nucleic Acids Res, 2009. 37:e123], with minor modifications. Following purification, mRNA was primed with Olig(dT)s and random hexamers, and reverse-transcribed to first-strand cDNA. Residual dNTPs were removed from the first-strand cDNA using Illustra MicroSpin G-25 columns (GE Healthcare). In the second-strand synthesis reaction, dUTPs were used instead of dTTPs. The converted double-strand cDNA was fragmented using a Bioruptor Sonicator for 60 cycles of 30 sec on and 30 sec off. After end-repair and adenine base addition, the cleaved double-strand cDNA fragments were ligated to Pair-end Adaptor Oligo Mix (Illumina) and size-fractionated on a 2% high resolution agarose gel (Sigma). cDNA fragments of 200 (+25 bp,-25bp) were recovered, and incubated with uracil-N-glycosylase (UNG) to digest the second-strand cDNA. Purified single-strand cDNA was then used as template for 15 cycles of amplification using pair-end PCR primers (Illumina). The amplified products were separated on a 2% agarose gel and a band between 225-275 bp excised.
characteristics.characteristic: description
characteristics.text: embryo
characteristics.characteristic: developmental stage
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: DP3
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/CL_0000809
characteristics.characteristic: title