RNA Expression in the left lateral lobe of the liver in BALB/cJ and BALB/cByJ substrains of mice to identify candidate mechanisms for Oxycodone QTLs

We found BALB/cByJ and BALB/cJ mice differ in their responses to oxycodone state dependent conditioned place preference, whole brain [oxycodone] and its metabolites [noroxycodone] and [oxymorphone] following oxycodone administration, the 53.5°C hot plate, mechanical stimulation in the von Frey test, and gross brain weight. We then identified a quantitative trait locus (QTL) on chromosome 15 for whole brain [Oxymorphone] (LOD = 7.07; p < 0.001), 13 for hot plate sensitivity (LOD = 10.7; p < 0.001; peak = 56 Mb) and a QTL for brain weight on chromosome 5 (LOD = 8.7; p < 0.001). These tissues were used to investigate the transcriptome of the liver in parental strain mice in order to generate hypothosis concerning the quantitative trait mechanism for our observed effects. Overall design: 16 BALB/cJ (8 females, 8 males) and 16 BALB/cByJ mice (8 females, 8 males) were trained and tested for state-dependent OXY-CPP using 1.25 mg/kg OXY (i.p.) as described above and sacrificed immediately after day 9 testing (30 min post-OXY) by rapid decapitation. The lateral left lobe of the liver was collected and preserved in RNAlater. RNA was extracted in RNAlater-preserved tissue using Trizol (Qiagen), ethanol precipitation, filtering columns (Qiagen), DNAse digestion (Qiagen), and elution with RNAse and nucleotide free water (Yazdani et al., 2015) and diluted to 100 ng/ul. RNA library preparation (poly-A selection) and RNA-seq were conducted at the University of Chicago Genomics Facility on an Illumina NovaSEQ6000 using a NovaSEQ SP-100 bp flowcell/reagent cassette. We used the R/Bioconductor package “scruff” to conduct demultiplexing, read alignment, read counting, quality checking and data visualization (Wang et al., 2019). Reads were trimmed for quality using Trimmomatic (Bolger et al., 2014). Trimmed reads were then aligned to the mm10 mouse reference genome (Ensembl) to generate BAM files for alignment using STAR (Dobin et al., 2013). For differential gene analysis in the spinal cord, the featureCounts read summarization program was used to count reads mapping to the “exon” feature in a GTF file obtained from Ensembl (GRCm38). Genes without 10 reads per million in at least 3 samples were excluded from analysis using EdgeR, and differential gene expression analysis of normalized counts was conducted using an appropriate design matrix, and reported using the topTable function.