title: Hippoglossus hippoglossus transcriptome from the Skin stage 7
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 (Einarsdottir et al., 2005). In brief, fertilised 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 darkeness at 5 oC until absorption of the yolk sack when they were moved to 100 l, round polyethylene start-feeding tanks containing sea-water at 10-11 oC under constant light conditions. The larvae were fed live artemia (Olsen et al., 1999) twice daily. Dead larvae were siphoned from the tanks each day and the mortality in each tank registered. 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 −20ºC. All handling procedures followed European guidelines (86/609/EU). Larvae were dissected into head, GI-tract and skin at standard development stages before, during and after metamorphic climax (n=6 per stage). Total RNA was extracted from all tissue/stages using a Maxwell®16 System (Promega, Madison, USA) and following the manufacturer’s instructions. Total RNA integrity was verified with an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, USA) and only the samples with RIN values equal to, or above 8 were used.    cDNA libraries were prepared and sequenced at the Max Planck Genome Centre (Cologne, Germany), using 5 µg of total RNA obtained from a pool of 6 samples for each tissue/stage. First, the whole transcriptome was enriched by depletion of the ribosomal RNA (rRNA, 28S, 18S, 5.8S and 5S) using a RiboMinusTM Eukaryote Kit (Life Technologies, Carlsbad, USA) following the manufacturer’s instructions. Total RNA (after rRNA depletion), was used to construct sixteen cDNA libraries (head from stage 5; head, skin and GI-tract from stages 7, 8, and 9, Sæle et al., 2004; stage 9 samples were split into 3 groups, 9A, 9B and 9C to differentiate by eye position) using a cDNA Rapid Library Preparation Kit (Roche 454 Life Sciences, Branford, USA) following the manufacturer’s instructions. Each library had a unique barcode and was amplified by emulsion PCR and sequenced on the GS-FLX platform (Roche 454 Life Sciences, Branford, USA).