WTCHG_31164_115
Mus musculus
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.
ENA-FIRST-PUBLIC
2013-10-31T17:01:07Z
ENA-LAST-UPDATE
2018-03-08T16:48:10Z
External Id
SAMEA2224742
INSDC center name
University of Oxford
INSDC first public
2013-10-31T17:01:07Z
INSDC last update
2018-03-08T16:48:10Z
INSDC status
public
Submitter Id
E-MTAB-1998:WTCHG_31164_115
broker name
ArrayExpress
common name
house mouse
developmental stage
adult
genotype
H2-ab1+/-
individual
AB68
maternal genotype
H2-ab1+/-
organism part
lung
paternal genotype
wild type
sample name
E-MTAB-1998:WTCHG_31164_115
scientific_name
Mus musculus
sex
female
specimen with known storage state
frozen specimen
strain
C57BL/6J x B6(C)-H2bm12/KhEgJ