Superscript 3 rnase transcriptase

Total RNA was extracted from sexually undifferentiated gonads (mean SL ∼4 cm) at 133 dph (T1) and sexually differentiated juvenile testis at 337 dph (T2).
A classical chloroform-isopropanol-ethanol RNA extraction protocol after a Trizol (Live Technologies, Scotland, UK) homogenization was used. RNA quality and concentration were measured by a ND-1000 spectrophotometer (NanoDrop Technologies) and checked on a 1% agarose/formaldehyde gel. RNA integrity was measured by a Bioanalyzer 2100 (RNA 6000 Nano LabChip kit Agilent, Spain). Samples with a 100–200 ng/µl RNA concentration and RIN>7 were used for microarray hybridizations.
In parallel, 200 ng of total RNA were treated by E. coli DNAse H and retrotranscribed (100 ng) to cDNA using SuperScript III RNase Transcriptase (Invitrogen, Spain) and Random hexamer (Invitrogen, Spain) following manufacturer's instructions.

Individual brain samples were separately weighed and homogenized with Tri-Reagent following manufacturer’s instructions (1 mL/100 mg of tissue; Molecular Research Centre, Sigma-Aldrich, UK). Total RNA was extracted from individual fish brains for both S. salar (Proactive = 88; Reactive =40, Figure S1a and Table S1) and D. labrax (Proactive = 20; Reactive = 20. Figure S1b and Table S1) using the standard TriReagent (Sigma-Aldrich, UK) based method following manufacturer’s instructions. The concentration of each sample (total RNA) was quantified by Nanodrop (ND-1000) and quality visualized under UV light in a 1% agarose gel containing 1 μg/ml ethidium bromide. 2 μg of total RNA was taken from each individual to synthesize cDNA with SuperScript III RNase Transcriptase (Invitrogen) and oligo-dT primer (Promega).

Different cDNA synthesis protocols were used depending on the purpose of the samples, following the manufacturer’s instructions of each kit. The Illumina RNA-seq cDNA library was prepared using 1 µg of RNA from each sample and TruSeq V2 kits (Illumina, CA, USA), with reduced RNA fragmentation time (3 min) to maximize obtention of longer reads. For PacBio IsoSeq, 1 µg of RNA per sample was used for first-strand synthesis by SMARTer PCR cDNA Synthesis Kit (Clontech). For quantitative RT-PCR (qPCR), 1 µg of total RNA per sample was used to synthesize cDNA with SuperScript III RNase Transcriptase (Invitrogen) and Oligo-dT primer (Invitrogen).