Automated High-Throughput RNA Sequencing

RNA was sequenced at Northwest Genomics Center, where next-generation sequencing libraries were prepared from 1.25 µg of total RNA in a high-throughput format, using the TruSeq Stranded mRNA kit (Illumina, San Diego, CA, USA). All the steps required for sequence library construction were automated and performed on a Sciclone NGSx Workstation (Perkin Elmer, Waltham, MA, USA). During library construction, rRNA was depleted by means of a poly-A enrichment, and first and second strand cDNA syntheses were performed. Each library was uniquely barcoded using Illumina adapters and amplified by PCR. After amplification and cleanup, library concentrations were quantified using the Quant-it dsDNA assay (Life Technologies, Carlsbad, CA, USA). Final libraries were normalized and pooled based on Agilent 2100 Bioanalyzer results (Agilent Technologies, Santa Clara, CA, USA) and size selected using a Pippin Prep (Sage Science, Beverly, MA, USA). Pooled libraries were diluted to a final concentration of 2–3 nM for sequencing on a HiSeq 4000, to a read depth of 30 million base pairs. Samples were multiplexed and sequenced on a HiSeq 4000. Lane-level sequencing reads were base quality checked using the FASTX-toolkit and FastQC, and aligned to hg19 with a reference transcriptome Ensembl v67, using TopHat2 suite [56 (link)] followed by matefixing, as described previously [28 (link)].