Illumina RNA-Seq Library Preparation

Samples with RNA integrity values of ≥7.0 by Agilent Bioanalyzer (Santa Clara, CA) were processed by the Mayo Clinic Medical Genome Facility Gene Expression Core into TruSeq libraries according to the manufacturer’s instructions (RNA Prep Kit v2, Illumina, San Diego, CA) and published elsewhere [25 (link)]. Paired-end DNA adaptors (Illumina) with a single “T” base overhang at the 3’ end were immediately ligated to the ‘A tailed’ cDNA population. Unique indexes, included in the standard TruSeq Kits (12-Set A and 12-Set B), were incorporated at the adaptor ligation step for multiplex sample loading on the flow cells. Libraries (8–10 pM) were loaded onto paired-end flow cells to generate cluster densities of 700,000/mm² following Illumina’s standard protocol for cBot and cBot Paired end cluster kit version 3. The flow cells were sequenced as 51 X 2 paired-end reads on an Illumina HiSeq 2000 using TruSeq SBS sequencing kit version 3 and HCS v2.0.12data collection software. Base-calling was performed using RTA version 1.17.21.3.