cDNA Library Preparation and Sequencing

The cDNA library was constructed using a modification of the cDNA Rapid Library Preparation Kit (Roche Hellas). In brief, 2 μg total RNA from each sample was fragmented by adding 2 μl fragmentation solution in a total volume of 18 μl, vortexed and incubated at 72°C for 30 s. Precipitation was performed at -20°C overnight in a total volume of 500 μl with 50 μl NaOAc, 2.5 ml EtOH and 1 μl glycogen (Invitrogen/VWR, Tromsø, Norway). First strand cDNA was synthesized using 5’-TTTTTTCTTGTTTTCTTTTCTTV-3’ primer [29 (link)]. Second strand synthesis as well as library preparation was constructed following the instructions of GSFLX cDNA rapid library protocol. Library quantification and quality control was performed using Quantiflur ST Fluorometer (SB Biotechnology Suppliers S.A.) and Agilent 2100 Bioanalyzer respectively. Next generation sequencing was performed according to GSFLX Titanium protocols.
Raw sequences have been submitted to Sequence Read Archive (SRA) division of the Genbank repository and can be access through the SRA web site under accession number SRX297084, SRX297965, SRX297966, SRX297967.