NAc punches were taken from individual mice (at least 8 animals/treatment group). Total RNA was prepared using TRIzol (Invitrogen) (Eipper-Mains et al., 2011). Average yield of total RNA per NAc punch was 6.3±0.6 µg. Total RNA used for NAc library preparation consisted of equal amounts of RNA from 8 individual mice for each treatment. One Saline, one Cocaine, and one 7-day Withdrawal mRNA-Seq library was prepared from each pooled RNA sample according to manufacturer’s specifications (Illumina). Briefly, total RNA was treated with DNase I; poly(A)+ RNA purified using Dynal magnetic beads (Invitrogen) was fragmented by partial alkaline hydrolysis (Ambion) and reverse-transcribed using random primers and SuperScript II (Invitrogen). cDNA was size-selected (~200 bp insert) on 2% agarose gels. Libraries were prepared for sequencing using the Paired-End DNA Sample Prep Kit (Illumina). NAc libraries were sequenced in nine lanes (3 technical replicates per sample) on an Illumina GAIIx using a 37-cycle paired-end sequencing protocol. Replicates were analyzed for intra-sample disparity and read data from all three lanes were then merged into one composite data file per sample; intra-sample coefficient of determination, R2 ≥ 0.98. The composite file was used for subsequent analyses.
