characteristics.text: 2012-05-04T17:00:33Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2018-03-08T16:04:02Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA1466715
characteristics.characteristic: External Id
characteristics.text: EMBL Genomics Core Facility
characteristics.characteristic: INSDC center name
characteristics.text: 2012-05-04T17:00:33Z
characteristics.characteristic: INSDC first public
characteristics.text: 2018-03-08T16:04:02Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS099181
characteristics.characteristic: SRA accession
characteristics.text: C57BL/6
characteristics.characteristic: StrainOrLine
characteristics.text: E-MTAB-997:Mbd3_KO_R1
characteristics.characteristic: Submitter Id
characteristics.text: cell_line
characteristics.characteristic: bio source type
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: embryonic stem
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/CL_0002322
characteristics.characteristic: cell type
characteristics.text: house mouse
characteristics.characteristic: common name
characteristics.text: Protocols: ES cells were cultured in 2i defined medium, a 1:1 mixture of DMEM/F-12 and Neurobasal with N2 and B27 supplements, 1uM PD0325901 and 3uM CHIRON99021. Total RNA was extracted using the TRIzol method followed by treatment with TURBO DNase (Ambion). Polyadenylated transcripts were selected from 2 µg total RNA using Sera-Mag beads (Thermo Scientific). Between 60 and 100 ng mRNA was then sheared to approximately 200 nt fragments by focused ultrasound on the Covaris S2 using the following parameters: Duty Cycle =10%, Intensity = 5, Cycles Per Burst = 200, for 75s with frequency sweeping enabled. First-strand cDNA synthesis was performed at 50°C for 2 hours using SuperScript III (Invitrogen) and random hexamer primers, followed by second-strand synthesis with DNA Polymerase I at 16°C for 2 hours in the presence of RNaseH. End repair of double-stranded cDNA products was carried out with T4 DNA polymerase and T4 polynucleotide kinase (New England Biolabs). Blunted, phosphorylated cDNA fragments were then 3'-adenylated via Klenow fragment and ligated to sequencing adapters (Illumina) by T4 DNA ligase at 20°C for 30 minutes. PCR amplification of library constructs was carried out with Phusion DNA polymerase (Finnzymes) for 13 cycles. Purification of reaction products between each step was performed with Ampure XP paramagnetic beads (Beckman Coulter). Prior to sequencing, the molarity and size distribution of the libraries was assessed by DNA 1000 microfluidic chips on the Agilent 2100 Bioanalyzer.
characteristics.characteristic: description
characteristics.text: normal
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/PATO_0000461
characteristics.characteristic: disease state
characteristics.text: Mbd3-/-
characteristics.characteristic: genotype
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: Mbd3_KO_R1
characteristics.characteristic: title