organism: Salmo salar
description: Protocols: Liver samples were collected immediately after dissection and placed in RNAlater. Liver slice samples were transferred immediately from culture media to a tube containing RNAlater, three slices per tube. Tubes with RNAlater and samples were allowed to sit at room temperature for 30 minutes before being stored at -20°C. Atlantic salmon were treated according to the Norwegian Animal Research Authority (NARA). Immediately after euthanization, liver was removed from saltwater stage (~200 g) fish fed Marine oil based, high EPA/DHA diet and samples were taken from the right lobe for sampling and liver slice culture. Liver slices were prepared immediately after dissection. Liver was removed from the fish and placed in a petri dish containing ice cold Hank's balanced salt solution (HBSS, Thermofisher). Liver was cut into 4x4x8mm strips, then superglued onto a plastic piston and encased in ultra-low melt agarose (Merck) at 27°C. Agarose was immediately solidified by contact with an ice cold metal cooling block. Encased liver was inserted into a compresstome VF-300 (Precisionary Instruments) and sliced into 300µm sections. Slices were collected in ice cold HBSS before being transferred to 15°C L15 media (Thermofisher) containing 1% penicillin-streptomycin (Thermofisher) and 5% fetal bovine serum (Merck) in 12 well cell culture plates with netwell inserts (Corning, 500µm membrane size). Liver slices were incubated at 15°C for nine days, refreshing media every three days. After three days slices were supplemented with either 70µM alpha-linolenic acid solubilized methyl-beta-cyclodextrin (BCD) at a 1:10 molar ratio (experimental), or 700µM empty BCD (control). Samples were taken on day 0 (before slicing), and days 1, 3, 4, 5, 6, 7, 8, and 9. 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 TrueSeq 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: 2018-11-06T16:18:17Z
attributes.tag: External Id
attributes.value: SAMEA5066844
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: 2018-11-06T16:18:17Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-7364:TC_D4_ALA-2
attributes.tag: age
attributes.value: 44
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: Atlantic salmon
attributes.tag: developmental stage
attributes.value: saltwater stage
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: growth condition
attributes.value: precise cut liver slice culture
attributes.tag: individual
attributes.value: fish 1
attributes.tag: organism part
attributes.value: liver
attributes.tag: sample name
attributes.value: E-MTAB-7364:TC_D4_ALA-2
attributes.tag: scientific_name
attributes.value: Salmo salar
attributes.tag: sex
attributes.value: not available
attributes.tag: strain
attributes.value: wild type