cDNA library preparation for RNA-Seq

The total RNA input used for sample preparation was 700 ng-1,500ng total RNA per sample. PolyA-enriched mRNA was purified from total RNA input using the Dynabeads mRNA-purification kit (Invitrogen, #61006) according to the manufacturer`s protocol, and eluted in a final volume of 50 µL. Ribosomal RNA (rRNA) contamination was assessed by Agilent 2100 Bioanalyzer (Total RNA protocol) according to manufacturer`s instructions. Samples with rRNA content >25% were subjected to a second round of mRNA purification and re-evaluated by Bioanalyzer as previously. The total volume of purified mRNA was adjusted to 81 µL with nuclease-free water, 9 µL of fragmentation solution (RNA fragmentation kit; Ambion, #AM8740) was added and samples were kept on ice. Fragmentation was carried out at 75°C for 3 min in a thermoblock, at 3 min samples were immediately cooled on ice and 11 µL of stop solution (RNA fragmentation kit, Ambion) was added. Cleanup was performed using the RNeasy Kit and RNA was eluted in 30 µL. Fragment size distribution and concentration was analysed by Bioanalyzer (mRNA protocol).

First strand cDNA synthesis was carried out with the SuperScript VILO Kit (Invitrogen, #11754050) in a 50 µL reaction with an additional 7.5 µg random hexamers (Invitrogen, #48190011) added to the reaction to increase cDNA yield. Reactions were incubated for 10 Min at 25°C (priming), 60 Min at 42°C (reverse transcription), and 5 Min at 85°C (termination). Reaction clean-up was carried out by gel filtration using Sephadex G50-columns (Mini Quick Spin DNA Columns; Roche #11814419001) according to the manufacturer’s protocol. Second-strand cDNA was synthesised in a final reaction containing the first strand cDNA reaction and final concentrations of: 550 µM of each dATP, cGTP, dCTP (Thermo Scientific, #R0181), and dUTP (deoxy-UTP sodium salt; Roche, #11934554001); 0.300 U DNA Polymerase I (E.coli; Invitrogen, #18010–025), 0.073 U E. coli DNA ligase (Invitrogen, #18052–019), and 0.015 U RNase H (Promega, #M4281); 20 µL 5X second strand buffer (Invitrogen, #10812–014), in a final volume of 100 µL. Reactions were incubated for 2 hr at 16°C then purified with by the MiniElute Reaction clean-up kit (Qiagen, #28004), eluted in 2 × 10 µL volumes in a single reaction tube. Samples were stored at −20°C prior to final library preparation for sequencing.