characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2016-03-01T17:02:04Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2016-02-15T12:26:26Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA3872865
characteristics.characteristic: External Id
characteristics.text: University of Cambridge
characteristics.characteristic: INSDC center name
characteristics.text: 2016-03-01T17:02:04Z
characteristics.characteristic: INSDC first public
characteristics.text: 2016-02-15T12:26:26Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS1059999
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-4461:HNES3_R2
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: HNES3
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: Human naive embryonic stem (HNES) cells were propagated in modified N2B27 medium supplemented with 1 uM PD0325901 (prepared in house), 1 uM CHIR99021 (prepared in house), 2.5 uM Go6983 (Sigma-Aldrich), 10 uM rho-associated kinase inhibitor (Y-27632, Calbiochem), 10 ng/ml human LIF (prepared in house) and 250 uM ascorbic acid (Sigma). N2B27 medium (1L) comprised 490 mL DMEM/F12 (Life Technologies), 490 mL Neurobasal (Life Technologies), 10 mL B27 (Life Technologies), 5 mL N2 (prepared in house), 10 ug/mL insulin (Sigma), 2 mM L-glutamine (Life Technologies) and 0.1 mM 2-mercaptoethanol (Sigma). N2 contains 100 ug/ml apo-transferrin (eBioscience, ABC2553), 3 uM sodium selenite (Sigma), 1.6 mg/mL putrescine (Sigma) and 2 ug/mL progesterone (Sigma) in DMEM/F-12 (Life Technologies). Cells were cultured in 5% O2, 7% CO2 in a humidified incubator at 37 C. Feeder cells were depleted by culture on gelatin-coated dishes for 40 minutes and harvesting cells in suspension. Total RNA was extracted with the TRIzol/chloroform method (Invitrogen), followed by re-suspension in RNAsecure (Ambion), incubation with TURBO DNase (Ambion) at 37 C for 1 hour, and 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 5 ug of total RNA using Ribo-Zero capture probes (Epicentre). RNA samples were sheared by ultrasonication on a Covaris S2 for 90 s 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 2 hours in the presence of 6 ug/mL actinomycin D (Sigma) to inhibit formation of second-strand products (Perocchi et al. Nucleic Acids Res 35:e128). Second-strand cDNA was synthesized by DNA Polymerase I in the presence of RNase H with dUTPs substituted for dTTPs at 16 C for 2 hours. Sequential end repair and 30-adenylation of cDNA products were 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). Products were ligated to barcoded adapters (NEXTflex-96, Bioo Scientific) by T4 DNA ligase (New England BioLabs) at 20 C for 30 min. Second-strand DNA was digested with uracil DNA glycosylase (UDG) and Endonuclease VIII at 37 C for 30 min. PCR amplification of first-strand library constructs was carried out with KAPA HiFi DNA polymerase (KAPA Biosystems) for 13 cycles using denaturing and annealing conditions optimized for even A/T versus G/C processing (Aird et al. Genome Biol 12:R18). Purification of reaction products at each step was performed with Ampure XP paramagnetic beads (Beckman Coulter). Library size distribution and molarity were assessed by DNA 1000 micrifluidic chips on the 2100 Bioanalyzer (Agilent).
characteristics.characteristic: description
characteristics.text: embryo stage
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: embryo
characteristics.characteristic: organism part
characteristics.text: Homo sapiens
characteristics.characteristic: scientific_name
characteristics.text: female
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/PATO_0000383
characteristics.characteristic: sex
characteristics.text: HNES3_R2
characteristics.characteristic: title