Infection, sampling and sample selection: Atlantic salmon in the BKD group were injected with 200 µl of R. salmoninarum to obtain final concentration of 2 × 10^8 colony-forming units (CFU) per fish. Fish in the control group were injected with 200 µl of sterile KDM-2 medium. After 13 days of infection, fish were euthanized using overdose of Benzocaine, and the head kidney of the each individual was sampled for transcriptome analyses. The collected samples were stored in RNAlater at 4°C for 24 h. Then, RNAlater was removed, and samples were kept at -80 °C until RNA extraction. Using a TaqMan qPCR assay, fish (n=5) with no infection from Control group as well as fish showing high (H-BKD) or low (L-BKD) infection level (i.e. expression level of R. salmoninarum 16S ribosomal RNA) from the BKD group were selected for microarray analyses. Total RNA was extracted from TRIzol-lysed samples, following the manufacturer’s instructions. Total RNA samples were on-column DNase-treated and column-purified using PureLinkTM RNA Mini Kit (Thermo Fisher Scientific) and PureLinkTM DNAse set (Thermo Fisher Scientific), according to the manufacturer’s protocol. The high-purity RNA samples (e.g. A260/230 and A260/280 ratios > 1.8) with tight 18S and 28S ribosomal RNA bands were used for the microarray analysis.
Label
Cy5
Label protocol
Five individuals per group (5 control, 5 L-BKD and 5 H-BKD; 15 samples in total) were used for microarray experiment. The microarray experiment was conducted using a common reference design. The mRNA of each sample was amplified and then labelled using 1 µg of DNase-treated and column-purified RNA and Amino Allyl MessageAmp™ II aRNA Amplification kit (Ambion®, Carlsbad, CA, USA), according to the kit’s procedure. A common reference was prepared using a pool of all 15 samples (15 µg aRNA from each sample) involved in the microarray experiment. The experimental samples (Control, L-BKD and H-BKD) were labelled with Cy5 (GE Healthcare, Buckinghamshire, UK), whereas the common reference samples were labelled with Cy3 (GE Healthcare), based on the manufacturer’s protocol.
Channel 2
Source name
pool of RNAs from all samples involved in the microarray experiment
Infection, sampling and sample selection: Atlantic salmon in the BKD group were injected with 200 µl of R. salmoninarum to obtain final concentration of 2 × 10^8 colony-forming units (CFU) per fish. Fish in the control group were injected with 200 µl of sterile KDM-2 medium. After 13 days of infection, fish were euthanized using overdose of Benzocaine, and the head kidney of the each individual was sampled for transcriptome analyses. The collected samples were stored in RNAlater at 4°C for 24 h. Then, RNAlater was removed, and samples were kept at -80 °C until RNA extraction. Using a TaqMan qPCR assay, fish (n=5) with no infection from Control group as well as fish showing high (H-BKD) or low (L-BKD) infection level (i.e. expression level of R. salmoninarum 16S ribosomal RNA) from the BKD group were selected for microarray analyses. Total RNA was extracted from TRIzol-lysed samples, following the manufacturer’s instructions. Total RNA samples were on-column DNase-treated and column-purified using PureLinkTM RNA Mini Kit (Thermo Fisher Scientific) and PureLinkTM DNAse set (Thermo Fisher Scientific), according to the manufacturer’s protocol. The high-purity RNA samples (e.g. A260/230 and A260/280 ratios > 1.8) with tight 18S and 28S ribosomal RNA bands were used for the microarray analysis.
Label
Cy3
Label protocol
Five individuals per group (5 control, 5 L-BKD and 5 H-BKD; 15 samples in total) were used for microarray experiment. The microarray experiment was conducted using a common reference design. The mRNA of each sample was amplified and then labelled using 1 µg of DNase-treated and column-purified RNA and Amino Allyl MessageAmp™ II aRNA Amplification kit (Ambion®, Carlsbad, CA, USA), according to the kit’s procedure. A common reference was prepared using a pool of all 15 samples (15 µg aRNA from each sample) involved in the microarray experiment. The experimental samples (Control, L-BKD and H-BKD) were labelled with Cy5 (GE Healthcare, Buckinghamshire, UK), whereas the common reference samples were labelled with Cy3 (GE Healthcare), based on the manufacturer’s protocol.
Hybridization protocol
The labeled aRNA (i.e. 825 ng) of each experimental sample was mixed with an equal amount of labeled aRNA of the common reference, and the resulting pool was fragmented based on the manufacturer's instructions (Agilent, Mississauga, ON). Each labelled aRNA pool (i.e. an individual sample and common reference) was co-hybridized to a cGRASP-designed Agilent 44K salmonid oligonucleotide microarray (GEO accession # GPL11299). The arrays were hybridized at 65°C for 17 h with 10 rpm rotation in an Agilent hybridization oven. The array slides were washed immediately following the manufacturer's instructions.
Scan protocol
The microarray slides were scanned at 5 µm resolution using SureScan Microarray Scanner System (Agilent Technologies, Santa Clara, CA) and Microarray Scan Control Software following the Agilent HD 2-color gene expression microarrays scan protocol.
Data processing
Agilent Feature Extraction Software v12.0 (Agilent Technologies) was used to extract and Loess-normalize the signal intensity data. Thereafter, using GeneSpring Software v14.9 (Agilent Technologies), the data were quality-checked so that probes with low and marginal quality as well as absent values in more than 25% of all 15 arrays were removed from the dataset.
