nucleic acid sequencing protocol
Hundred base pair fragments were sequenced in paired-end on a Genome Analyzer- GAIIx (Illumina). This technical replication aimed at testing the effect of two distinct insert (fragment) sizes on the quality of the assembly: does a mixture of two insert sizes produce a better assembly than each size separately.
nucleic acid library construction protocol
Total RNA from liver and brain was isolated using TRIzol (Invitrogen, Carlsbad, CA). Samples were treated with the DNA-free kit (Invitrogen, Saint-Aubin, France) using the manufacturers protocol. Total RNA was measured with a Nanodrop2000 (Thermo Scientific, Villebon-sur-Yvette, France). Total RNA pools of brain and liver were used for RNA-seq. Double stranded cDNA preparation and sequencing were performed at the BioPuces Strasbourg Department of the Institut de Genetique et de Biologie Moleculaire et Cellulaire (IGBMC Strasbourg, France). Sample preparation was performed using the Illumina mRNA sample preparation kit (Cat.No. RS-930-1001), involving the following steps. Poly-A containing mRNA were isolated from 4 ug of total RNA. The mRNA was then fragmented using divalent cations under elevated temperature (incubation time was reduced in order to obtain longer fragments). The cleaved RNA fragments were reverse transcribed into first stranded cDNA using random primers, second strand cDNA was then synthesized. Adaptors were ligated to the end-repaired cDNA. At the purification step, we selected two size ranges of templates: a pool of 350 and a pool of 500 base pair (bp) long. From then on, these two banks were treated separately for downstream enrichment by PCR and for paired-end RNA-seq. One flow cell was dedicated to each organ, and to each fragment size, i.e., a total of four flow cells (half a run) were employed.
Seabasses were sacrificed at midnight and midday. Brain and livers were collected and were stored in RNAlater (Invitrogen, Saint-Aubin, France) at 4C overnight. RNAlater was then removed and samples were stored at -80C until use.