Sample 25
Salmo salar
Protocols: During sampling, randomly selected fish (76 in total) were placed in an anaesthetic bath (~20 g of MS-222/150 L) for 5-10 min, followed by macroscopic examination (gross morphology) of gill tissue as per routine procedure to monitor fish welfare indicators. This procedure is performed by fish health professionals, using semi-quantitative 6-grade scoring systems for PGD, from 0 (no visual pathology) to 5 (severe visual pathology) (Gill Health Initiative, 2015; Bloecher et al., 2018). Each of the 8 gill arches (4 on left and 4 on right side) was scored separately, generating in total 8 PGD scores per fish. Although scoring for PGD is typically non-destructive and requires only light anaesthesia (e.g., 10 g of MS-222/150 L), fish in our study were subjected to terminal anaesthesia for subsequent harvesting of tissue samples. Immediately after PGD scoring, fish were bled and the gill arch with the highest PGD score was excised for both transcriptome profiling and histopathological examination. For transcriptomic profiling, 3 transverse sections (gill arch ~3 mm long with 3-4 pairs of filaments) from dorsal, medial and ventral regions of the gill (Figure 1) were transferred to RNAlater® (Sigma-Aldrich, St. Louis, MO, USA), kept at 4°C overnight for equilibration and then stored at −80°C prior to RNA extraction. All remaining tissue from the same gill arch was transferred to freshly prepared seawater Davidson's fixative (Cadoret et al., 2013) for 24 h, followed by a short-term exposure to 10% neutral buffered formalin before tissue processing for histopathological examination. Total RNA extraction was performed on individual gill transverse sections (45 gills x 3 sections) after removing the arch tissue and leaving only full-length filaments for further processing. Briefly, the RNA was isolated by homogenization of ~100 mg of gill tissue in TRIzol® Reagent (Ambion by Life Technologies, Carlsbad, CA, USA), using 3 mm tungsten carbide beads and a TissueLyser II Disruption System (Qiagen GmbH, Hilden, Germany). Following isolation, the RNA was quantified by spectrophotometry (NanoDrop Technologies, Wilmington, DE, USA) and its integrity was confirmed by electrophoresis (Agilent Technologies, Santa Clara, CA, USA). The three individual RNA samples that originated from the same gill were then pooled to generate a single RNA sample per gill tissue (n = 45 RNA pools in total), with an equimolar contribution of RNA from dorsal, medial and ventral regions of the gill to each pool. All but one pooled gill RNA samples had a 260/280 ratio > 1.8 and RIN number > 9.3, thus meeting the criteria for RNA-sequencing. The sample with degraded RNA was eliminated from further processing. RNA-seq library preparation and sequencing were carried out by Edinburgh Genomics at the University of Edinburgh (UK). The libraries for each of the 44 samples were constructed using the TruSeq Stranded mRNA Sample Preparation Kit (Illumina, San Diego, CA), according to the manufacturer's instructions.
ENA-FIRST-PUBLIC
2020-06-01T04:05:54Z
ENA-LAST-UPDATE
2020-03-06T11:48:56Z
External Id
SAMEA6607915
INSDC center name
UOA
INSDC first public
2020-06-01T04:05:54Z
INSDC last update
2020-03-06T11:48:56Z
INSDC status
public
PGD score
1
Submitter Id
E-MTAB-8855:Sample 25
age
2
broker name
ArrayExpress
common name
Atlantic salmon
developmental stage
mature
fish id
39
genotype
outbred
organism part
gill filament
sample name
E-MTAB-8855:Sample 25
scientific_name
Salmo salar
site
B
strain
Fanad