GSM9218953: SP_FD_D2_S68; Salmo salar; RNA-Seq
Total RNA was isolated from head kidney and spleen from 10 fish per dietary group at each location. Briefly, a 5 mm stainless steel bead and 900 μL QIAzol lysis reagent (Qiagen) were added to 50 mg of each sample. Then, a TissueLyser bead mill (Qiagen) at 20 Hz for 2 min was used (twice) to lyse the samples. The homogenate was recovered and 180 μL chloroform were added before vortexing and centrifugation at 14,000 x g (4 °C for 15 min). Each upper phase was recovered in a new tube and 450 μL isopropanol were added. After gentle manual shaking and incubation at room temperature, the samples were centrifuged again (14,000 x g at 4 °C for 15 min). The supernatants were removed and 200 μL 70% ethanol were added before centrifugation (14,000 x g at 4 °C for 10 min). The pellets were left to dry for 5-10 min at room temperature. Finally, each pellet was resuspended in 40 μL RNAse-free water and incubated for 10 min incubation at 55 °C. The RNA concentration of the samples was estimated with a NanoDrop™ 8000 spectrophotometer (Thermo Fisher Scientific) according to instructions from the manufacturer. Then, RNA integrity (RIN values) was measured with an Agilent 4150 TapeStation (Agilent) following the supplier's protocols. All samples had RIN values ≥ 7. Novogene (United Kingdom) performed the libraries and RNA-seq through an Illumina NovaSeq 6000 System (150 bp paired-end RNA-seq and 30 million reads depth).