Strand-Specific RNA-Seq Library Preparation

mRNA was fragmented into 50- to 200-nucleotide fragments by incubation in RNA fragmentation reagent (Ambion) for 15 min. Strand-specific cDNA libraries were prepared by the method of Parkhomchuk et al. (34 (link)). Briefly, the fragmented mRNA was then converted into double-stranded cDNA using the SuperScript double-Stranded cDNA synthesis kit (Invitrogen), per the manufacturer’s instructions with the following changes. After the first strand synthesis, the products were purified using Illustra Microspin G-50 columns to remove all the deoxynucleoside triphosphates (dNTPs), and a dUTP mixture containing dUTP, in lieu of dTTP, was used for the second strand synthesis. Samples were sequenced by the University of Vermont Cancer Center Advanced Genome Technologies Core. Each sample was sequenced at a minimum of 1,000× coverage using 100-bp single-end reads on an Illumina HiSeq1000 instrument. Staphylococcus aureus was included as a control for strandedness.