Gene expression over a broad (106) dynamic range was evaluated in each library. Expression was quantified in reads per kilobase of exon per million mapped sequence reads (RPKM), a normalized measure of exonic read density (Mortazavi et al., 2008). For the three NAc libraries, pairwise scatter plots revealed similarly varied expression between the Saline/Cocaine, Saline/Withdrawal and Cocaine/Withdrawal libraries (Figure 1A–C). The variability of gene expression between samples was highest for genes expressed at lower levels. For this reason, a lower limit of expression was applied before examining regulation. Since biogenic amine transporter (Slc6a2, NET; Slc6a3, DAT; Slc6a4, SERT) transcripts are not expressed in the NAc (http://mouse.brain-map.org/), we used their RPKM values to set this lower limit; for inclusion in subsequent analyses, a gene had to have an RPKM of > 1.0 in at least one of the three treatment groups. We used qPCR to validate the RNA-Seq data (Figure 1D,E). The RNA-Seq and qPCR expression levels of 47 transcripts from five neurotransmitter systems from the Saline sample showed strong agreement across the two methods (Pearson’s r = 0.75) over a
