MC_D9_mBCDch_1
Salmo salar
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 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), 5% fetal bovine serum (Merck), and 20nM human insulin (Sigma) 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 either every day or every three days. After three days slices were supplemented with 70µM alpha-linolenic acid solubilized methyl-beta-cyclodextrin (BCD) at a 1:10 molar ratio or 700µM BCD. Samples were taken on days 0 (before slicing), 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.
ENA-FIRST-PUBLIC
2019-03-31T17:01:58Z
ENA-LAST-UPDATE
2018-11-07T15:50:14Z
External Id
SAMEA5071823
INSDC center name
NORWEGIAN UNIVERSITY OF LIFE SCIENCES
INSDC first public
2019-03-31T17:01:58Z
INSDC last update
2018-11-07T15:50:14Z
INSDC status
public
Submitter Id
E-MTAB-7367:MC_D9_mBCDch_1
age
25
broker name
ArrayExpress
common name
Atlantic salmon
developmental stage
freshwater stage
environmental history
media changed daily
genotype
wild type genotype
growth condition
precision cut liver slice culture
individual
fish 1
organism part
liver
sample name
E-MTAB-7367:MC_D9_mBCDch_1
scientific_name
Salmo salar
sex
not available
strain
wild type