RNAseq Atlantic salmon tongue - in vivo infection trial with Yersinia ruckeri (NVIO 10705)
RNAseq Atlantic salmon tongue - in vivo infection trial with Yersinia ruckeri (NVIO 10705)
Fish were euthanised with a lethal dose of Benzoak vet (ACD Pharmaceuticals AS, Norway). Tongue samples were preserved in RNAlater™ Stabilization Solution (Thermo Fisher Scientific, Lithuania), kept overnight at 4oC, and then transferred to -20oC until RNA isolation.    The infection trial was conducted in a flow-through system with 12 ppt salinity at 12°C. Two 500-L tanks were used: one for the control group and one for the infected group. Each tank was stocked with 30 unvaccinated fish (~100 g), which were acclimated to the system for one week. The bacterial suspension was prepared as outlined in section 2.4, and fish were infected by bath exposure to the pathogen for four hours at 8 x 107 CFU/mL. This concentration was previously identified in a routine infection model development at Nofima. The other fish group was handled similarly, but PBS was added instead, serving as the control group. During the exposure period, oxygenation was maintained above 85% saturation. Fish were transferred to new tanks after the exposure period. Four days post-infection, tongue samples were collected for RNA-Seq analysis (N=5, per control and infected group).  RNA of all samples was isolated using the Agencourt RNAdvance™ Tissue Total RNA Purification Kit (Beckman Coulter Inc., USA) using the Biomek 4000 automated workbench station (Beckman Coulter, Inc.). To provide an en masse representative of the organ, the entire tongue was homogenised first using ceramic beads in FastPrep-96™ (MP Biomedicals LLC, USA) for 2 mins before a small portion of the homogenate (ca 10 mg) was used for RNA isolation. RNA concentration and purity were assessed using a NanoDrop 8000 Spectrophotometer (Thermo Fisher Scientific). Samples designated for RNA sequencing underwent additional quality control using an Agilent 2100 Bioanalyzer (Agilent, California, USA). The RNA integrity number (RIN) values of all samples were above 8.0.  Total RNA samples were sent to the Norwegian Sequencing Centre (OUS, Norway). A strand-specific TruSeq RNA Library Prep Kit (Illumina, CA, USA) was used to prepare RNA-seq libraries using the manufacturer's protocol. 