organism: Salmo salar
description: 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.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2019-03-31T17:01:58Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-11-07T15:17:41Z
attributes.tag: External Id
attributes.value: SAMEA5071601
attributes.tag: INSDC center name
attributes.value: NORWEGIAN UNIVERSITY OF LIFE SCIENCES
attributes.tag: INSDC first public
attributes.value: 2019-03-31T17:01:58Z
attributes.tag: INSDC last update
attributes.value: 2018-11-07T15:17:41Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-7368:PC_D6_ALAH2O2_2
attributes.tag: age
attributes.value: 25
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell type
attributes.value: hepatocyte
attributes.tag: common name
attributes.value: Atlantic salmon
attributes.tag: developmental stage
attributes.value: freshwater stage
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: individual
attributes.value: fish 1
attributes.tag: organism part
attributes.value: liver
attributes.tag: sample name
attributes.value: E-MTAB-7368:PC_D6_ALAH2O2_2
attributes.tag: scientific_name
attributes.value: Salmo salar
attributes.tag: sex
attributes.value: not available
attributes.tag: strain
attributes.value: wild type