Tissue-Specific Transcriptomic Insights into Myxozoan Infections: Immune and Stress Responses in the Kidney and Head Cartilage of Rainbow Trout
Tissue-Specific Transcriptomic Insights into Myxozoan Infections: Immune and Stress Responses in the Kidney and Head Cartilage of Rainbow Trout
At 90 dpi, fish from each group were euthanized in buffered tricaine methanesulfonate (MS-222) overdose (250–500 mg/L) [81], and death was confirmed before collection of Kidney and Head cartilage sampling and stored at -80°C until further analysis. Total RNA was isolated from kidney and head cartilage (HC) collected at 90 dp. For each tissue, samples from 15 fish were processed (3 fish per group; 5 experimental groups in total), using a RNeasy Mini Kit (Qiagen, Hilden, Germany) following the manufacturer's protocol. An on-column DNase digestion step was included to remove residual genomic DNA. RNA quantity and purity were assessed with a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, Wilmington, USA), and integrity was checked by agarose gel electrophoresis. Only samples with RNA integrity numbers (RIN) ≥ 8.0 were used for library preparation. For each sample, 500 ng of total RNA were subjected to mRNA enrichment using the Lexogen Poly(A) RNA Selection Kit V1.5 (Lexogen, Vienna, Austria). mRNA-enriched fractions were then used to generate cDNA libraries with the CORALL mRNA-Seq Library Prep Kit (Lexogen), according to the manufacturer's instructions, yielding a total of 30 libraries (15 kidney and 15 HC). Library size distribution and quality were evaluated using a High Sensitivity D1000 ScreenTape Kit on the Agilent 4200 TapeStation System.