title: Illumina NovaSeq 6000 paired end sequencing; RNA-seq of polar cod (Boreogadus saida) liver slice culture exposed to benzo[a]pyrene and ethynylestradiol
design: RNA-seq of polar cod (Boreogadus saida) liver slice culture exposed to benzo[a]pyrene and ethynylestradiol
library_construction_protocol: The experiments were performed in August 2019 on the Norwegian Nansen Legacy project cruise to the Northern Barents Sea. Polar cod were collected by trawling (78.7°N, 34.0°E) and kept in a 500 L tank with running seawater at the ambient seawater temperature outdoor on the deck of the cruise ship in the Northern Barents Sea. Female polar cod (total 5 fish) with average body and liver weight (Mean  SD) of 35.8  7.8 and 3.2  1.2 g, respectively were used in this experiment. Polar cod PCLS preparation was performed by using protocols previously described for Atlantic cod (Eide et al., 2014; Yadetie et al., 2018). The liver was sliced using Leica vibrating blade microtome VT1200 (Leica, Wetzlar, Germany). Slices (250 μm thick strips, about 4 x 4 mm) were cultured in the culture medium (Leibowitz-15 medium, Life Technologies™ Gibco®, Paisley, UK) supplemented with 10% charcoal-stripped fetal bovine serum and 1% penicillin–streptomycin–amphotericin (10,000 U/mL potassium penicillin, 10,000 μg/mL streptomycin and 25 μg/mL amphotericin B; Sigma-Aldrich) in the presence of DMSO vehicle control, BaP alone (0.1, 1, and 10 µM), or in combination with low concentrations of EE2 (5 nM),  at 6°C with horizontal shaking (50 rpm) for 72 hours. Liver slices from a total of 5 female fish (biological replicates n = 5) were distributed in each group (paired sample design). After 72 hours in culture, slices were collected and snap-frozen in liquid nitrogen and stored at -80 °C until further processing. Total RNA was isolated from frozen slices using TRI Reagent according to the manufacturer's protocol (Sigma-Aldrich, St. Louis, MO, USA). RNA concentration and quality were assessed using NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). All the RNA samples had RNA integrity number (RIN) values > 9. Sequenced by Novogene on Illumina NovaSeq 6000 System
attributes.tag: Experimental Factor: compound
attributes.value: benzo(a)pyrene
attributes.tag: Experimental Factor: dose
attributes.value: 10
attributes.tag: Experimental Factor: replicate
attributes.value: 5