Naive S4 mice (N = 12/line/sex) were euthanized, brains removed and immediately frozen on dry ice. Frozen brains were sliced in 55-μ coronal sections on a freezing microtome at −13°C and slices containing the NAc were mounted on polyethylene naphthalate-covered 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 seconds each and then air-dried. The NAc shell was dissected bilaterally on a Leica LMD-6000 (Leica Microsystems Inc., Buffalo Grove, IL) using known anatomical landmarks (Paxinos & Franklin 2007). Dissected tissue was processed with the Arcturus Picopure Kit (ThermoFisher Scientific, Waltham, MA) yielding on average 200 ng of total RNA. RNA quality was assessed using the Caliper Labchip GX (PerkinElmer, Waltham, MA) and RNA Quality Scores (RQS).
