Barcoded libraries were pooled and sequenced on Illumina NextSeq system (Illumina, San Diego, CA, USA) producing about 500 M reads of non-paired 75-nt sequence. Up to 32 barcoded samples were pooled together producing on average 12 M reads per sample. RNAseq analysis was carried out using the BaseSpace platform from Illumina. The RNAseq-generated FASTQ files were aligned to the USCSrn5 Rattus norvegicus reference genome with STAR aligner [38] with allowed mismatches set to 14. Differentially expressed (DE) mRNAs were determined using the DeSeq2 package based on the negative binomial distribution and a false discovery rate of 0.1% [39]. In brief, paired RNAseq data for each transcript are compared using Wald testing which is a more powerful method than others to detect significant differences in low expression transcripts [40]; those with Wald p values < 0.05 are ordered, and an adjusted p value is then determined using Benjamini-Hochberg approach to minimize false discovery to 0.1% or less. This method does not take into consideration the magnitude of the difference in expression. Functional and pathway analysis were performed using DAVID [41] and GO Consortium [42, 43] platforms.
