We also assessed the mRNA expression of these risk genes in brains of two strains of rats (i.e., BN-Lx/ CubPrin and SHR/OlaPrin) using RNA-Seq technology. After ribosomal RNA depletion, the total RNAs were reversely transcribed to cDNAs that were then sequenced on the Illumina HiSeq2000 system. RNA-Seq data were available from the PhenoGen website (http://phenogen.ucdenver.edu) (16). After reads were trimmed for adapters and quality, they were aligned to the RGSC5.0/rn5 version of the rat genome using TopHat2 (17). RNA expression was quantitated for genes identified in a denovo genome-guided transcriptome reconstruction using CuffLinks and all uniquely aligned reads (18). Read fragments per kilobase of transcript per million reads (FPKM) for the candidate genes were calculated. To confirm these RNA-Seq results in rats, gene expression data were also generated using whole brain tissue of naïve mice using Affymetrix mouse whole genome oligonucleotide microarrays (i.e., GeneChip Mouse Genome 430 v2.0 Array, Affymetrix, Santa Clara, CA) (16). A BXD recombinant inbred panel of 30 strains (parental strains are C57BL/6J & DBA/2J) was assessed and the microarray data were normalized using MAS5 method. When a specific mRNA detected by RNA-Seq in rats was present in >90% of the mouse samples, this expression was confirmed.
