Cdna rapid library preparation kit

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.

Total RNA was isolated from ~ 100 mg of leaf and root tissue of 12 individual F. picrosperma and 3 individual F. venosa using the Qiagen mini plant kit (Hilden, Germany), according to the manufacturer’s protocol. The initial yield and purity of RNA were measured using a Nanodrop spectrophotometer 2000c (Thermo Scientific, Waltham, MA, USA) at 260 and 280 nm and agarose gel electrophoresis. An Agilent Bioanalyzer 2100 (Agilent Technologies, USA) was used to analyse the RNA integrity number (RIN). High-quality total RNA (RIN > 7) was provided to Novogene (Beijing, China) for cDNA synthesis (cDNA Rapid Library Preparation Kit, Roche, Mannheim, Germany) and paired-end Illumina HiSeq 2500 sequencing (Illumina, San Diego, CA, USA).

cDNA libraries of the Atlantic halibut skin, GI-tract and head were prepared from pools of 6 samples per stage to obtain 5 μg of total RNA. Ribosomal RNA was depleted using RiboMinus™ Eukaryote Kit (Life Technologies, Carlsbad, USA) and following the manufacturer’s instructions. A cDNA Rapid Library Preparation Kit (Roche Life Sciences, USA) was used to construct sixteen cDNA libraries; head from stage 5 and head, skin and GI-tract from stages 7, 8 and 9A, 9B and 9C. Each library had a unique barcode and was amplified by emulsion PCR and sequenced on a GS-FLX platform (Roche Life Sciences, USA) following the manufacturers recommendations. The sequencing assigned quality scores are available at the NCBI Short Read Archive (SRA; Accession number: SRP044664).