Sybr safe

The reagents and solvents were used without further purification. The ligand 1-(3-chloro-2-hydroxypropyl)-2-methyl-5-nitroimidazole (onz) was obtained from Sigma Aldrich, as well as thionyl chloride, sulfadiazine, Hoechst 33258, cacodylic acid, (3-(N-morpholino) propanesulfonic acid) (MOPS), ascorbic acid, hydrogen peroxide, SYTOX^® Green, SYBR™ Safe and the calf thymus DNA. The metal salts: Na₂CO₃, Na₂SO₄, CuCl₂⋅2H₂O, CuBr₂, ZnCl₂ and ZnBr₂, and solvents: acetonitrile, ethyl acetate, hexane, ethanol, dimethyl sulfoxide, and 1-octanol were obtained from J.T. Baker. NaCl was purchased from Fisher, TBE 10X from Invitrogen, sodium salt of calf thymus DNA (ct-DNA, type I fibrous) from Sigma-Aldrich, plasmid pBR322 (4361 bp, 0.25 mg mL⁻¹) from Thermo Scientific, agarose from Ecogen and ethidium bromide 10 mg mL⁻¹ from Promega.

For each timepoint (control, 7 dpc and 14 dpc), 4 resistant and 4 susceptible fish, representing 8 different families, were selected; fish were classified in resistant / susceptible based on their individual EBVs and family mortalities, as previously described [15 (link)]. Gills, head kidney and spleen RNA samples from the same fish were extracted from preserved tissue samples in TRI reagent (Sigma, UK) and RNA extracted following the manufacturer’s instructions (n = 24 per tissue; control = 8; 7 dpc = 8; 14 dpc = 8). The RNA pellet was eluted in 15 μL of nuclease-free water and quantified on a Nanodrop 1000 spectrophotometer (NanoDrop Technologies) prior to DNAse treatment with QuantiTect® Reverse Transcription kit (Qiagen). The quality of the RNA was examined by electrophoresis on a 1% agarose gel (Sigma Aldrich), prepared in Tris-Acetate-EDTA (TAE) buffer, stained with 1% SYBR Safe (Sigma Aldrich) and run at 80 V for 30 min. Sample concentration was measured with Invitrogen Qubit 3.0 Fluorometer using the Qubit RNA HS Assay Kit (ThermoFisher Scientific). The 3’mRNA tag-seq libraries were prepared by Oxford Genomic Centre using the poly-A tail as an adapter, incorporating priming site for 1st strand synthesis, followed by RNA template removal. The libraries were sequenced on a Illumina Novaseq6000 with an average of 13.1 M reads (minimum 9.3 M).

For each timepoint (control, 7 dpi and 14 dpi) 4 fish with high breeding values for resistance and 4 fish with low breeding values for resistance, representing 8 different families, were selected according to their estimated genomic breeding value for ISAV resistance. Heart RNA was extracted from preserved tissue samples (n = 24; 8 x controls, 8 × 7 dpi, 8 × 14 dpi) in TRI Reagent (Sigma, UK) and RNA extracted following the manufacturer’s instructions. The RNA pellet was eluted in 15 μL of nuclease-free water and quantified on a Nanodrop 1000 spectrophotometer (NanoDrop Technologies) prior to DNAse treatment with QuantiTect® Reverse Transcription kit (Qiagen). The quality of the RNA was examined by electrophoresis on a 1% agarose gel (Sigma Aldrich), prepared in Tris-Acetate-EDTA (TAE) buffer, stained with 1% SYBR Safe (Sigma Aldrich) and run at 80 V for 30 min. Sample concentration was measured with Invitrogen Qubit 3.0 Fluorometer using the Qubit RNA HS Assay Kit (ThermoFisher Scientific). PolyA RNA-Seq libraries were prepared using Illumina’s TruSeq RNA Library Prep Kit v2 by Oxford Genomic Centre, and sequenced on an Illumina Novaseq6000 as 150 bp paired-end reads yielding an average of 51 M reads per sample (minimum 38 M).