RNA sequencing and analysis were carried out as previously reported (McClintick et al., 2015). Briefly, strand-specific libraries were prepared after ribo-reduction using Life Technologies SOLiD™ Total RNAseq kit (Life Technology, Carlsbad, CA). Library preparations were done in balanced batches. All samples for each brain region were pooled in equal molarity before EZbead preparation, followed by sequencing on a combination of SOLiD4™ and SOLiD™ 5500xl sequencers (50 base reads and 75 base reads, respectively). Aliquots of the same library preparations were sequenced on both machines. An average of 22.7 M (vHip) and 24.1 M (mPFC) reads per sample were mapped to Rn4 (Table 1). The edge® package (Robinson, McCarthy, & Smyth, 2010) was used to identify genes differentially expressed between control and alcohol groups. FDR was calculated within edge® according to Benjamini and Hochberg (Benjamini & Hochberg, 1995). Analysis was limited to those genes with ≥ 1 count per million in at least three samples. Library preparation batch was included as a factor in the analysis.
