characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2013-10-31T17:01:07Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2018-03-08T16:48:10Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA2224691
characteristics.characteristic: External Id
characteristics.text: University of Oxford
characteristics.characteristic: INSDC center name
characteristics.text: 2013-10-31T17:01:07Z
characteristics.characteristic: INSDC first public
characteristics.text: 2018-03-08T16:48:10Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS361231
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-1998:WTCHG_34370_208
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: house mouse
characteristics.characteristic: common name
characteristics.text: Protocols: All mice were housed and procedures carried out at the WTCHG. Mice were maintained in a specific pathogen free facility in individually ventilated cages, and given food and water ad libitum. All experiments were carried out in accordance with U.K. national (Home Office) and institutional guidelines. Hippocampus and lung RNA was extracted from 30 mice from a reciprocal cross of C57BL/6J x B6(C)-H2bm12/KhEgJ. Total RNA was isolated from frozen lung using RNeasy(r) kit, and from frozen hippocampus using RNeasy(r) micro kit (Qiagen, Crawley, UK). RNA concentrations were determined using a NanoDrop spectrophotometer, and lung RNA quality, purity and integrity were assessed using an Agilent 2100 Bioanalyser (Agilent Technologies, Waldbronn, Germany). Hippocampus RNA quality was not routinely assessed on the Bioanalyser because the quantities were too low. Three or four mice from each combination of sex, F1 genotype and parental genotype were used. RNA was barcoded, pooled and sequenced on a lane of a Hiseq2000 using 51bp paired end reads. Total RNA quantity and integrity were assessed, using Quant-IT RiboGreen RNA Assay Kit (Invitrogen, Carlsbad, CA, USA) and Agilent Tapestation 2200 R6K. mRNA enrichment was achieved by processing 5ug of total RNA using the Magnetic mRNA Isolation Kit from NEB (S1550S) with minor modifications. Generation of double stranded cDNA and library construction were performed using NEBNext(r) Ultra(tm) Directional RNA Library Prep Kit for Illumina(r) (E7420L) according to manufacturer specifications. Upon ligation of Illumina Adapters (Multiplexing Sample Preparation Oliogonucleotide Kit) each library was size selected with two Ampure Bead bindings (firstly, 1:0.7x volume and secondly, the supernatant from the first bind was taken for a 1:1.7x volume clean-up). The following custom primers (25 uM each) were used for the PCR enrichment step: Multiplex PCR primer 1.0: 5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3' Index primer: 5'-CAAGCAGAAGACGGCATACGAGAT[INDEX]CAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3' Indices used the eight bases tags developed by WTCHG (Lamble et al. in press). Amplified libraries were analysed for size distribution using the Agilent Tapestation 2200 D1K. Libraries were quantified by quantitative RT-PCR using Agilent qPCR Library Quantification Kit and a MX3005P instrument (Agilent) and relative volumes were pooled accordingly. Finally, a second quantitative RT-PCR was performed to measure the relative concentration of the pool compared to a previously sequenced mRNA library in order to determine the volume to use for sequencing.
characteristics.characteristic: description
characteristics.text: adult
characteristics.characteristic: developmental stage
characteristics.text: H2-ab1+/-
characteristics.characteristic: genotype
characteristics.text: AB55
characteristics.characteristic: individual
characteristics.text: H2-ab1+/-
characteristics.characteristic: maternal 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: hippocampus
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/UBERON_0002421
characteristics.characteristic: organism part
characteristics.text: wild type
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0005168
characteristics.characteristic: paternal genotype
characteristics.text: Mus musculus
characteristics.characteristic: scientific_name
characteristics.text: female
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/PATO_0000383
characteristics.characteristic: sex
characteristics.text: frozen specimen
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/OBI_0000922
characteristics.characteristic: specimen with known storage state
characteristics.text: C57BL/6J x B6(C)-H2bm12/KhEgJ
characteristics.characteristic: strain
characteristics.text: WTCHG_34370_208
characteristics.characteristic: title