Illumina HiSeq 2500 sequencing; RNA-seq of Atlantic salmon primary hepatocytes treated with alpha-linolenic acid
RNA-seq of Atlantic salmon primary hepatocytes treated with alpha-linolenic acid
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.
Experimental Factor: time
8
Experimental Factor: compound
alpha-linolenic acid (70 micromolar) and hydrogen peroxide (100 micromolar)