Following sequencing, reads from each library were aligned to a reference transcriptome using Bowtie (Langmead et al., 2009). For libraries generated from tissue samples taken from the F0 mice, the reference was either the C57BL/6J or CAST/EiJ reference transcriptome, as appropriate. For the F1 mice, we aligned reads to a reference that contained both the C57BL/6J and the CAST/EiJ transcriptomes. In all cases, MMSEQ (Turro et al., 2011) was used to estimate gene expression levels and, in the case of the F1 samples, to estimate allele specific gene expression levels. We normalised the expression estimates for the F0 data using the approach of Anders and Huber (Anders and Huber, 2010). We defined genes as expressed using the following criteria. A gene is defined as expressed in the F0 mice if the expression estimate in at least one of the 12 F0 mice is >= 10, and as expressed in both the F0 and F1 mice if the F0 criterion is satisfied and, additionally, the estimate is >= 10 across both alleles of the gene in at least one of the 6 F1i and in one of the 6 F1r mice.
Directional double-stranded cDNA was generated using the Superscript Double-Stranded cDNA Synthesis kit (Invitrogen), with Uracil substituted for Thymine in the second strand. 250ng of double-stranded cDNA was fragmented prior to library preparation by sonication in the Diagenode Bioruptor set on Hi for 5 minutes of 30 seconds on/off cycles, repeated twice; each sonicated cDNA sample was then treated to generate blunt ended double-stranded DNA. Following end repair, libraries were treated to add an overhanging Adenine nucleotide and Illumina paired-end (PE) adapters were ligated on both ends of each clone in the library. Strand-specificity was introduced by digesting the second strand of cDNA using a uracil-specific enzyme. Each library was subsequently amplified using PCR with Illumina???s PE primers. Size selection was performed by gel electrophoresis and 200-300 base pair fragments were extracted from a 2% agarose gel.
All mice used in this study were housed and handled in accordance with the Animals (Scientific Procedures) Act 1986. Mice were housed and cared for in the Cambridge Research Institute Biological Resource Unit with a twelve hour light/dark cycle, and were provided with chow food from Lab Diet plus water ad libitum. Mice were sacrificed by cervical dislocation at 4-6 months of age, between the hours of 9:30am and 11:30am. The liver was then cut and the mouse perfused with PBS by injection into the heart. Subsequently, the liver was removed and the gall bladder and hepatic vein cut away. A sample of approximately 50mg-100mg was taken from a single liver lobe and frozen on liquid nitrogen for storage at -80C until use.
For each liver sample, total RNA was extracted using the RNeasy Mini kit (Qiagen 74104) as per manufacturer???s instructions. Before further processing, each total RNA sample was treated to digest contaminating DNA. From each sample, polyadenylated mRNA was enriched from the total RNA using the PolyATract mRNA isolation system.