V1
Library construction procedures were performed at the National Center for Genome Resources in Santa Fe, New Mexico, USA. Magnetic RNA Purification Beads (Dynabeads: Invitrogen) were used to isolate the Poly-A RNA which was then fragmented by incubation at 95°C for 8 min. RT-PCR for first strand cDNA synthesis from the isolated Poly-A RNA was performed using SuperScript II enzyme (Intvitrogen), First Strand Master Mix (Illumina), and random hexamer primers (Illumina). Double-stranded cDNA was then synthesized using Second Strand Master Mix (Illumina). Double-stranded cDNA was purified by Agencourt AMPure XP beads (Beckman Coulter). Blunt end cDNA was prepared by removing 3’ overhangs and filling in 5’ overhangs using End Repair Mix (Illumina). 3’ ends were then adendylated using A-Tailing Mix (Illumina). Universal and barcoded TruSeq Adapters (Illumina) were ligated to cDNA ends. Polymerase Chain Reaction (PCR) was conducted with KAPA HiFi HotStart ReadyMix (KAPA Biosystems) and PCR Primer Cocktail (Illumina) to selectively amplify the cDNA fragments that had TruSeq adapters ligated to both ends. PCR products were purified using Agencourt AMPure XP beads. PCR product quality was assessed using a 2100 Bioanalyzer and cDNA quantity was measured by Nanodrop ND-1000 (Thermo Scientific). Samples were normalized to 10nM equimolar concentrations and pooled prior to flow cell injection.