title: Illumina HiSeq 2500 sequencing; RNA-seq of precision cut liver slice (PCLS) culture before slicing and up to 9 days after slicing, changing media either every day or every 3 days, with insulin
design: RNA-seq of precision cut liver slice (PCLS) culture before slicing and up to 9 days after slicing, changing media either every day or every 3 days, with insulin
library_construction_protocol: 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.
attributes.tag: Experimental Factor: growth condition
attributes.value: precision cut liver slice culture
attributes.tag: Experimental Factor: environmental history
attributes.value: media changed daily
attributes.tag: Experimental Factor: time
attributes.value: 8
attributes.tag: Experimental Factor: compound
attributes.value: methyl beta-cyclodextrin