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 PAG was micropunched from 6.04 mm to 7.30 mm post bregma, using procedures previously described (McBride et al., 2014). Other brain regions of these animals have been studied (McBride et al., 2014, McClintick et al., 2015). 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 8.4 for the samples.
