characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2014-09-10T17:02:19Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2018-03-08T21:12:10Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA2738358
characteristics.characteristic: External Id
characteristics.text: European Molecular Biology Laboratory
characteristics.characteristic: INSDC center name
characteristics.text: 2014-09-10T17:02:19Z
characteristics.characteristic: INSDC first public
characteristics.text: 2018-03-08T21:12:10Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS537888
characteristics.characteristic: SRA accession
characteristics.text: E_MTAB-2857:H9_R1
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: H9
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0003045
characteristics.characteristic: cell line
characteristics.text: pluripotent stem cell
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/CL_0002248
characteristics.characteristic: cell type
characteristics.text: human
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_9606
characteristics.characteristic: common name
characteristics.text: Protocols: Conventional human pluripotent stem cell (PSC) medium (FGF/KSR) comprised DMEM/F12 (Sigma-Aldrich) with 10ng/ml bFGF (prepared in-house) and 20% KSR (Invitrogen) supplemented with 100mM 2-mercaptoethanol (Sigma-Aldrich, cat. M7522), MEM non-essential amino acids (Invitrogen, cat. 11140050) and 2 mM L-glutamine (Invitrogen, cat. 25030024). Cultures were passaged every five to seven days as small clumps by dissociation with 0.025% Trypsin, 1mg/ml Collagenase IV (Invitrogen17104-019), KSR (final 20%) and 1mM CaCl2. Cells were maintained on irradiated mouse embryonic fibroblasts (MEF) in 5% O2. Total RNA was extracted with the TRIzol/chloroform method (Invitrogen), followed by resuspension in RNAsecure (Ambion), incubation with TURBO DNase (Ambion) at 37 C for 1 hour, further phenol/chloroform extraction and ethanol precipitation. RNA integrity was assessed with the RNA 6000 Nano assay on the 2100 Bioanalyzer (Agilent). Ribosomal RNA was depleted from 5ug of total RNA using Ribo-Zero capture probes (Epicentre). RNA samples were sheared by ultrasonication on a Covaris S2 for 90s set at Duty Cycle 10, Cycles per Burst 200 and Intensity 5. Fragmented RNA was reverse-transcribed with a combination of random hexamer and oligo-dT primers (New England Biolabs) by SuperScript III (Invitrogen) at 50 C for 2h in the presence of 6ug/ml actinomycin D (Sigma) to inhibit second-strand products. Second-strand cDNA was synthesized by DNA Polymerase I in the presence of RNase H with dUTPs substituted for dTTPs at 16 C for 2h. Sequential end repair and 3'-adenylation of cDNA products was carried out with T4 DNA polymerase and T4 polynucleotide kinase (20 C), and with exo- Klenow fragment (65 C) in the presence of dATPs (New England BioLabs). These were ligated to barcoded adapters (NEXTflex-96, Bioo Scientific) by T4 DNA ligase (New England BioLabs) at 20 C for 30min. Second-strand DNA was digested with uracil DNA glycosylase (UDG) and Endonuclease VIII at 37 C for 30min. PCR amplification of first-strand library constructs was carried out with KAPA HiFi DNA polymerase (Kapa Biosystems) for 13 cycles. Purification of reaction products at each step was performed with Ampure XP paramagnetic beads (Beckman Coulter). Library size distribution and molarity was assessed by the DNA 1000 assay on the 2100 Bioanalyzer (Agilent).
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: fresh specimen
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/OBI_0000971
characteristics.characteristic: specimen with known storage state
characteristics.text: H9_R1
characteristics.characteristic: title