title: Hippoglossus hippoglossus transcriptome from whole larvae stage 5
design: Atlantic halibut larvae were obtained from the aquaculture company Fiskeldi Eyjafjarðar Ltd. (Iceland) in December 2009. Larvae were reared in full-sea water using standard commercial procedures and normal metamorphosis was observed. In brief, fertilized eggs from several spawning batches were hatched in an open system of egg incubators. Yolk sack larvae were transferred to silo-shaped (10 m3) through-flow systems in complete darkness at 5 °C until absorption of the yolk sack when they were moved to 100 L, round polyethylene start-feeding tanks containing sea-water at 10-11 °C under constant light conditions. The larvae were fed live Artemia twice daily. The larvae were euthanized with a lethal dose of MS-222 (50 mg.l-1, ethyl 3-aminobenzoate methanesulfonate salt, Sigma-Aldrich, St. Louis, USA). Photographs were taken of each larvae and developmental staging was performed using mytome height and standard length (defined in Sæle et al., 2004) and then stored individually in RNAlater (Life Technologies, Carlsbad, USA) at −20°C. All handling procedures followed European guidelines (86/609/EU). Total RNA was extracted from whole individuals (n = 5 per stage) using a Maxwell®16 System (Promega, Madison, USA) following the manufacturer’s instructions. RNA integrity and concentration was verified with an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, USA) and only samples with RIN values equal to, or above 8 were used. SOLiD sequencing was carried out on cDNA libraries constructed using SOLiDTM Total RNA-Seq Kit (Applied Biosystems, CA). Ribosomal RNA was depleted from total RNA of whole individual Atlantic halibut extracted as described above. A pool of RNA from 3 individuals was used to prepare stage 5 library. Each library had a unique barcode and cDNA was purified to eliminate contaminants, amplified by emulsion PCR and sequenced in two different lanes on a SOLiD AB 5500xl Genetic Analyser platform (Applied Biosystems, USA).