normalization data transformation protocol
The read counts per Ensembl gene were computed by Tophat and written to text files (one per tissue, H2-ab1.hip.gene.counts.txt, H2-ab1.lung.gene.counts.txt) where the rows are genes and columns are individual mice.
high throughput sequence alignment protocol
After standard quality control reads were aligned to the mouse reference genome (mm9) using Tophat (Trapnell, Pachter, & Salzberg, 2009) with default parameters.
nucleic acid sequencing protocol
Sequencing was performed as 51bp paired end reads on a HiSeq2000 according to Illumina specifications.
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.
nucleic acid extraction protocol
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.
nucleic acid library construction protocol
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.