This study incorporated prefrontal cortex tissue from 27 BXD strains, nucleus accumbens and ventral midbrain tissue from 35 BXD strains, as well as B6 and D2 tissue from all three regions. Frozen tissue for a given brain region and strain was pooled from 4–5 animals and homogenized with Aurum™ total RNA fatty and fibrous tissue extraction kit (BioRad, catalog #732–6830) and a Tekmar homogenizer. RNA concentration was determined by absorbance at 260 nm, and RNA quality was analyzed by electrophoresis on a Experion analyzer (BioRad, Hercules, CA) and 260/280 absorbance ratios. All RNA samples had RNA quality indices (RQI)≥8. Total RNA (5 µg) derived from each pool and spike-in poly-A RNA controls were reverse transcribed into double-stranded cDNA using Affymetrix SuperScript® one-cycle cDNA kit (Invitrogen, catalog #A10752030). Biotin-labeled cRNA was synthesized from cDNA using the GeneChip IVT labeling kit (Affymetrix, part #900449) according to manufacturer's instructions, purified using the RNAeasy Mini Kit (Qiagen, Mountain View, CA), and quantified by absorbance at 260 nm. Labeled cRNA samples were hybridized to Mouse Genome 430 2.0 microarrays (Affymetrix, part #900497) according to the
