Differential expression gene analysis
The output from FeatureCounts was imported into DESeq2 (version ‘1.32.0’) (Love et al., 2014) in R (R version 4.1.1) (Team, 2013) for normalization and differentially expressed genes analysis.
qPCR Primer/probe optimization and cDNA synthesis
Primer and probe optimization : Fifty transcripts (genes) that were significantly DE between antibiotic and probiotic treatments versus the control treatment in the DESeq2 analysis were selected for printing on OpenArray Taqman qPCR chips (Supplementary Table S2). Four endogenous control genes (β-2-microglobulin, β-Actin, ribosomal protein L13, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)) were selected from previous studies (Geffroy et al., 2021; Limbu et al., 2018; Toews et al., 2019) to normalise the transcription profiles of the candidate transcripts Primers for the candidate transcripts were designed using Geneious Software v7.1.5 (http://www.geneious.com) and optimized on DNA from Chinook salmon fry. After PCR optimization, the primers were tested on a subset of our cDNA samples with SyBr® Green Dye I (Thermo Fisher Scientific) following the manufacturer’s protocol on the QuantStudio 12K Flex Real‐Time PCR System (Thermo Fisher Scientific). After testing positive for amplification of the expected sized fragment using SyBr® Green assays, new qPCR primers and Taqman® probes were developed using Primer Express® Software v3.0.1 (Thermo Fisher Scientific) for all 54 genes (50 candidate and 4 control genes; Supplementary Table 1). The qPCR primers spanned intron‐exon boundaries with a short amplicon size (50–100 bp). The Taqman® probe was designed for a melting temperature between 57 and 60 °C.
cDNA synthesis : RNA was quality tested on a random subset of the samples both on a 2100 Bioanalyzer and on 2% agarose gels. RNA Integrity Number (RIN) values were consistent among samples, ranging between 7 and 9.8, while gel images showed the expected rRNA bands. The RNA concentration for each sample was estimated by Spark® multimode microplate reader and NanoQuant Plate™ (Tecan, Morrisville, NC, USA). All total RNA preparations had purity values of 1.8 – 2.1 (A260/A280) with concentrations ranging from 2,000 to 5,000 ng/μL. TURBO DNA-free™ Kits (Thermo Fisher Scientific, cat. no. AM1907) were used to remove genomic DNA contamination. Total RNA was converted to cDNA using High Capacity cDNA Kits (Applied Biosystems, Ontario, Canada), following the manufacturer’s protocol. Reverse transcriptase reactions contained 10 µL of total RNA at a concentration of 200 ng/μL, 2 µL of 10X RT random primers (Applied Biosystems), 0.8 µL of dNTP (100mM), 50 U of MultiScribe RT (Applied Biosystems) and 40 U of RNase Inhibitor (Applied Biosystems) in a 2 µL of 10X RT buffer at a final volume of 20 µL. RT reactions were incubated at 25°C for 10 min followed by 37°C for 120 min and were stopped by incubating at 85°C for 5 min. cDNA samples were stored at –20°C until further analysis.