Tissue collection, RNA isolation, library preparation and sequencing
After each fish was sacrificed, gill samples were immediately collected using sterilized tweezers and stored in RNAlater ® (Ambion® RNA, Life Technologies™). Of the 8 fish that were exposed in each experimental group, a total of three fish from each control (SwC and FwC) and a total of five fish from each exposure group (SwFw and FwSw) were processed for sequencing. For these 16 samples, messenger RNA (mRNA) was isolated from the gill tissue from each sample separately, using the mRNA direct kit with dynabeads (Invitrogen) as described by the manufacturer. The mRNA concentration and purity were quantified using an RNA 6000 Pico Kit on an Agilent 2100 Bioanalyzer (BioRad) according to the protocol, and all samples were diluted down to 0.125 µg/µL before cDNA synthesis. Libraries for RNAseq were prepared using the TrueSegTMRNA low-throughput protocol (Illumina), where all samples were fragmented for four minutes to obtain the required size distribution. All libraries were sequenced as 100bp paired-end at the Norwegian Sequencing Centre on the Illumina HiSeq 2000.