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: SAMEA2738357
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: ERS537887
characteristics.characteristic: SRA accession
characteristics.text: E_MTAB-2857:mES_PDL_R2
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: E14tg2A
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0006275
characteristics.characteristic: cell line
characteristics.text: embryonic stem cell
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/CL_0002322
characteristics.characteristic: cell type
characteristics.text: house mouse
characteristics.characteristic: common name
characteristics.text: Protocols: Cells were cultured in N2B27 medium (Ndiff227, StemCells Inc) supplemented with 1uM PD0325901 (PD03) and murine LIF (prepared in-house). 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: Mus musculus
characteristics.characteristic: organism
characteristics.text: Mus musculus
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_10090
characteristics.characteristic: organism
characteristics.text: Mus musculus
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: 129/Ola
characteristics.characteristic: strain
characteristics.text: mES_PDL_R2
characteristics.characteristic: title