RNA-seq library preparation protocol

First strand synthesis of rRNA-depleted RNA was performed using the Super Script III kit (Life Technologies) and random priming using random hexamers. Second-strand synthesis was performed with dUTP in place of dTTP to enable strand-specific sequencing (Parkhomchuk et al. 2009 (link)). Samples were end repaired, A-tailed, and ligated to NEXTflex DNA Barcodes for multiplex sequencing. Adapter dimers were removed using AMPure beads (Agencourt). Samples were then treated with UNG and amplified using 10 cycles of PCR prior to sequencing. Samples were sequenced using an Illumina 2000 single-end 50-bp run.