PC_D6_mBCD_1
Salmo salar
Protocols: Samples were taken for sequencing on days 5, 6, and 8. Atlantic salmon were treated according to the Norwegian Animal Research Authority (NARA). Immediately after euthanization, liver was removed and samples were taken from the right lobe for sampling and liver slice culture. After euthanization, the liver was removed and rinsed in ice cold Mg2+/Ca2+ free HBSS, before ~100 ml of the same buffer was injected with a 50 ml syringe and 27G needle, at various places to wash out blood cells. Then, 30 ml of HBSS with 150U/ml Collagenase (Sigma) was injected, before the tissue was finely chopped. The tissue suspension was incubated for 1h at 10-12°C with agitation. Dissociated cells were collected by cell straining (70 µm) and centrifugation for 10 min at 100g. After three washes in HBSS, the pellet was dissolved in base media supplemented with 10 µM insulin (Merck) and grown at 200k density at 15°C. Cells were treated on day 5 with 70µM alpha-linolenic acid (ALA) solubilized in methyl-beta-cyclodextrin (BCD) at a 1:10 molar ratio, 700µM empty BCD, or 70µM ALA and 100µM hydrogen peroxide. Samples were taken for sequencing on days 5, 6, and 8. mRNA was isolated using the RNeasy universal kit (QIAGEN). All RNA samples were eluted in a final volume of 30µl and eluate was run through the column twice to maximize yield. RNA concentration was determined on a Nanodrop 8000 and RNA quality was determined on a Agilent 2100 bioanalyzer using Agilent RNA 6000 nano chips. All RNA samples used for sequencing had a RIN of at least 7. Libraries were constructed using the Illumina TruSeq Stranded mRNA protocol. Concentration and mean length were determined by running cDNA libraries on a bioanalyzer 2100 using Agilent DNA 1000 chips.
ENA-FIRST-PUBLIC
2019-03-31T17:01:58Z
ENA-LAST-UPDATE
2018-11-07T15:17:42Z
External Id
SAMEA5071605
INSDC center name
NORWEGIAN UNIVERSITY OF LIFE SCIENCES
INSDC first public
2019-03-31T17:01:58Z
INSDC last update
2018-11-07T15:17:42Z
INSDC status
public
Submitter Id
E-MTAB-7368:PC_D6_mBCD_1
age
25
broker name
ArrayExpress
cell type
hepatocyte
common name
Atlantic salmon
developmental stage
freshwater stage
genotype
wild type genotype
individual
fish 1
organism part
liver
sample name
E-MTAB-7368:PC_D6_mBCD_1
scientific_name
Salmo salar
sex
not available
strain
wild type