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.