GSE196334
RNA Expression QTL mapping in an F2 cross of BALB/cJ and BALB/cByJ mice to identify candidate genes for behavioral/metabolic 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 (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 for expression QTL mapping to highlight the candidate genes Zhx2 (57 Mb) as the top transcript with the strongest association at the ch15 oxymorphone locus and identified H2afy (56 Mb) as the top transcript with the strongest association at the ch13 hot plate locus. Overall design: 64 BALBcJ x BALBcByJ F2 mice (32M, 32F) were used for eQTL mapping and were 78–127 days old on day 1 of testing. Mice were trained using a CPP protocol with either saline (i.p.) or 1.25 mg/kg OXY (i.p.) (Kirkpatrick & Bryant, 2015) and were then sacrificed by rapid decapitation on the final day of experimental testing, 30 min after receiving either saline (i.p.) or OXY (1.25 mg/kg, i.p.). Using a brain matrix, striatal and hippocampal tissues were dissected. Striatal punches were harvested at bregma 1.5 - -0.5mm and sampled with a 2 mm punch. Hippocampal tissues were dissected from bregma --0.5 - -2.5 using a sterile metal spatula to peel away the cortical layer. Tissues were 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 51, and differential gene expression analysis of normalized counts was conducted using an appropriate design matrix, and reported using the topTable function.