At 8 weeks of age, naive S4 mice, balanced for sex and line, were euthanized, the brains removed and immediately frozen on dry ice. Frozen brains were sliced in 55 micron coronal sections on a freezing microtome at −13°C and slices containing the nucleus accumbens, the amygdala, and the medial prefrontal cortex were mounted on PEN slides. Mounted slices were lightly thionin-stained under RNAse-free conditions and dehydrated in increasing concentrations of ethanol diluted in RNAse free water (50, 70, 95, and 100%) for 30 s each and then air-dried. The shell of the accumbens (SH), the CeA and the PL were dissected bilaterally on a Leica LMD-6000 using known anatomical landmarks (Franklin and Paxinos, 2008). Dissected tissue was processed with the ARCTURUS PicoPure kit. RNA quality was assessed using the Caliper LabChip GX and RNA Quality Scores (RQS). Only samples with RQS scores of >7 and >100 ng of total RNA were used for library formation. Sample numbers were as follows: SH-71; CeA-67; and PL-54. For reasons that were not clear, the percentage of extractions from the PL for high quality RNA was significantly lower.
