RNA-seq Analysis of Nucleus Accumbens

The left nucleus accumbens from each rat was purified using the RNeasy kit using the manufacturer's directions. cDNA libraries were created by reverse transcribing the RNA and creating the second strand. Blunt ends were phosphorylated and “a-tailed” so that adapters could be ligated to both ends. RNA was sequenced with a HiSeq 1000 system from Illumina. Four samples were added to each lane. cDNA was amplified using “bridge” amplification. Base calls were made using fluorescently labeled nucleotides. Over 100 million reads were taken at 50 bp (paired-end reads). FastQC (v0.9.1) (Andrews, 2014 ) was used to check the quality of the reads. Reads were mapped to the rat reference genome (RN4) using Tophat2 (v2.0.4) (Kim et al., 2013 (link)) and Bowtie2 (v2.0.0.6) (Langmead and Salzberg, 2012 (link)) software packages. The R package EdgeR (v.3.0.8) (Anders and Huber, 2010 (link); Robinson et al., 2010 (link)) was then used for analysis using the “trimmed mean for M-values” (TMM) method for normalization and tag-wise dispersion using “count” data. A likelihood ratio F-test was used for generating P-values to compare EC vs. IC rats. Transcripts significantly regulated (p < 0.05) were overlaid onto the IPA energy metabolism pathway for comparison to protein values.