Instrument: Illumina Genome Analyzer IIx
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: Fish were euthanized with an overdose of Tricaine Methanosulfonate (250mg/L) dissolved in seawater. Livers were harvested and flash-frozen in liquid nitrogen at -196oC. These procedures followed an approved institutional IACUC protocol and all animals were treated humanely. RNA was extracted from liver samples using TRIzol reagent (Invitrogen, CA) and further purified using the RNeasy Mini kit with DNAse to remove DNA (Qiagen, Valencia, CA). RNA concentrations were determined at 260 nm using an ND1000 (Nanodrop, Wilmington, DE). RNA was tested for structural integrity with the 6000 Nano LabChip assay from Agilent, (Santa Clara, CA, USA). Only RNA samples with RIN scores > 7.0 were used for RNA-seq. Libraries for RNA sequencing (RNAseq) were generated using Illumina TruSeq™ RNA Sample Preparation Kit (Illumina, USA) in accordance with the manufacturer's recommendations (Figure 2A). The library fragments were purified with AMPure XP system (Beckman Coulter, USA) to select cDNA fragments of approximately 300 bp in length. DNA fragments with ligated adaptor molecules on both ends were selectively enriched using Illumina PCR Primer Cocktail in a 10-cycle PCR reaction. Products were purified using AMPure XP system and quantified using the Agilent high sensitivity DNA assay on the Agilent Bioanalyzer 2100 system.