RNA-Seq Library Preparation and Analysis

First-strand cDNA synthesis was performed with 195 ng total RNA using anchored oligo-dT and SuperScript III First-Strand Synthesis SuperMix (Thermo Fisher Scientific). Second strand cDNA synthesis was performed using RNase H, DNA polymerase I, and Second Strand Buffer (Thermo Fisher Scientific). Double-stranded cDNA was purified using DNA Clean & Concentrator-5 (Zymo, Irvine, CA). Library preparation was performed using the Nextera XT DNA Library Preparation Kit (Illumina, San Diego, CA). Libraries were cleaned using Agencourt AMPure XP beads according to manufacturer's instructions (Beckman Coulter). Libraries were evaluated by the High Sensitivity DNA Kit on the 2100 Bioanalyzer. They were then multiplexed and sequenced on an Illumina HiSeq with 50 bp paired-end reads. Reads were aligned to NCBI build 37.2 using Tophat (v2.1.0).²² (link) Cuffquant and Cuffnorm (Cufflinks v2.2.1) were used to quantify expression levels and calculate normalized fragments per kilobase of transcript per million mapped reads (FPKM) values.²³ (link)