title: Illumina NovaSeq 6000 paired end sequencing; RNA-seq of Atlantic salmon olfactory rosette and gill
design: RNA-seq of Atlantic salmon olfactory rosette and gill
library_construction_protocol: The samples were collected from a fish trial described and reported earlier*. The trial conducted experimental induction of amoebic gill disease and thereafter treatment with peracetic acid of Atlantic salmon smolts weighing 80-90 g. The samples were collected from the group that was not infected and treated. Briefly, the fish were reared in 800 L tank operating in a flow through system under the following conditions: water flow rate in the tanks was 6-7 L/min, the water temperature at 14.5°C, oxygen at >85% saturation, salinity at 35 ppt, photoperiod set at 24 light: 0 darkness, and continuous feeding regime with a commercial diet (Skretting Nutra Olympic 3 mm, Averøy, Norway) administered through a belt feeder. Six fish with almost identical weight (82 ± 5.5 g) were dip netted from the tank and humanely euthanised with a bath overdose of Benzoak Vet (ACD Pharmaceuticals AS, Leknes, Norway). Gills from the second gill arch and olfactory rosette, both from the left side, were dissected and suspended in RNAlater (Ambion©, Connecticut, USA), kept at room temperature for 12 h for penetration and afterwards stored at -80°C until RNA isolation. *Lazado, D.A. Strand, M.W. Breiland, F. Furtado, G. Timmerhaus, M.C. Gjessing, S. Hytterød, G. Merkin, L.-F. Pedersen, K. Pittman, Mucosal immune and stress responses of Neoparamoeba perurans-infected Atlantic salmon (Salmo salar) treated with peracetic acid shed light on the host-parasite-oxidant interactions, Frontiers in Immunology, 13 (2022) 948897. Total RNA was isolated from the gills and olfactory rosette using the Agencourt RNAdvance™ Tissue Total RNA Purification Kit (Beckman Coulter Inc., CA, USA) with the aid of Biomek 4000 automated workbench station (Beckman Coulter, Inc., CA, United States). The purified RNA was quantified using NanoDrop 8000 Spectrophotometer (ThermoFisher Scientific, United States) and integrity was further evaluated using the Agilent® 2100 Bioanalyzer™ RNA 6000 Nano Kit (Agilent Technology Inc., Santa Clara, CA, USA). All samples had an RNA Integrity Number (RIN) above 8.0. Total RNA samples were sent to the Norwegian Sequencing Center (UiO, Norway). RNA libraries were prepared using strand specific TruSeq RNA Library Prep Kit (Illumina, CA, USA).