Brains were rapidly extracted and flash-frozen in isopentane in dry ice and stored at −80 C until sectioning. Brains were sectioned (300 μm) and the mPFC was micro-punched from +3.2 mm to +2.2 mm from bregma, including both prelimbic and infralimbic cortices, and the ventral hippocampus was micro-punched from −5.3 mm to −6.3 mm from bregma, using procedures previously described (McBride, Kimpel, et al., 2014). RNA was extracted using twice the suggested ratio of TRIzol (Life Technologies, Carlsbad, CA) to tissue (Edenberg et al., 2005), followed by additional purification using RNeasy columns (Qiagen, Hilden Germany). The yield, concentration and purity of the RNA were measured by Nanodrop (Thermo Fisher Scientific, Waltham, MA) spectrum from 220 nm to 340 nm. Quality was further assessed by Agilent Bioanalyzer (Agilent Technologies, Santa Clara, Ca); RNA integrity numbers (RIN) averaged 8.7 for vHip and 8.4 for mPFC samples. Gene expression changes have previously been reported in four brain regions of these same animals: nucleus accumbens shell and central nucleus of the amygdala (McBride, Kimpel, et al., 2014), dorsal raphe nucleus (McClintick et al., 2015) and periaqueductal gray (McClintick et al., 2016).
