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.