RNA-seq of Oncorhynchus tshawytscha, gill tissue
Libraries were constructed using NEBNext Ultra RNA Library Prep for Illumina kits (New England Biolabs kits E7490 and E7530), as per manufacturers instructions. Briefly, mRNA was enriched using oligo (dT) magnetic beads, fragmented, and reverse transcribed to cDNA. Samples were ligated with NEBNext multiplex oligo adaptor kits (New England Biolabs) for barcoding individual samples. Ligated DNA was then amplified by PCR. Libraries were quantified using a Qubit 2.0 fluorimeter and quality checked for insert size using a BioAnalyzer, then precisely quantified using KAPA Biosystems universal Illumina library quantification kit (Roche). Barcoded, prepared samples were pooled into two pools (with 10 and 14 individuals each) for sequencing on a HiSeq 4000 (Illumina) using paired-end (PE) 150 mode without PhiX with the aim to obtain approximately 10 million PE reads per sample.