RNA-seq Protocol for Rhesus Macaque Transcriptome

Five (5) nanograms of total RNA was used as input for cDNA synthesis using the Clontech SMART-Seq v4 Ultra Low Input RNA kit (Takara Bio) according to the manufacturer's instructions. Amplified cDNA was fragmented and appended with dual-indexed bar codes using the NexteraXT DNA Library Preparation kit (Illumina). Libraries were validated by capillary electrophoresis on an Agilent 4200 TapeStation, pooled at equimolar concentrations, and sequenced on an Illumina HiSeq3000 at 100SR, yielding 20–25 million reads per sample. Alignment was performed using STAR algorithm version 2.5.2b (Dobin et al., 2013 (link)). Transcripts were annotated using MacaM assembly and annotation of the Indian rhesus macaque genome (Zimin et al., 2014 (link)) (http://www.unmc.edu/rhesusgenechip/index.htm#NewRhesusGenome). Transcript abundance estimates were calculated internal to the STAR aligner using the algorithm of htseq-count generating the raw read count table (Anders et al., 2015 (link)).