BioProject

Purpose: The objective of this study is to use Atlantic cod (Gadus morhua) PCLS culture and RNA-seq analysis to map gene expression responses to BaP and EE2 exposure in Atlantic cod. Methods: A total of 8 (3 females and 5 males) juvenile cod (G. morhua) (average body weight 498 g) were used for PCLS (n = 6-8 per group). PCLS preparation and chemical exposure in the culture was performed as previously described (Eide et al., 2014). Cod liver slices were cultured in 24-well plates in the growth medium containing DMSO vehicle (CAS No: 67-68-5), BaP (CAS No: 50-32-8) (10 or 1000 nm), EE2 (CAS No: 57-63-6) (10 or 1000 nm) or BaP and EE2 mixture (10 or 1000 nm each). After 48 hors of culture, the slices were collected and snap frozen in liquid nitrogen and stored at -80 °C for RNA extraction. Results: In total, we had 47 high-quality samples, which were aligned to the published CDS of Atlantic cod (ftp://ftp.ensembl.org/pub/release-90/fasta/gadus_morhua/cds/Gadus_morhua.gad) using HISAT2 v2.1.0 (Kim et al., 2015). Counts were generated from the alignments using SAMtools v1.4.1 (Li et al., 2009). Differential expression analysis was performed using edgeR v3.18.1 (McCarthy et al., 2012) between control group and each treated group using paired test, after removing the genes with all-zero counts across all samples of the two compared groups. Differentially expressed genes were defined by p-value < 0.05 after adjustment using the Benjamini-Hochberg multiple testing correction. Several genes were differentially expressed in the BaP and EE2 and mixture treatment groups compared to the group. Hierarchical clustering showed that signature expression profile of top differentially expressed genes in response to the single treatment was distinctly maintained also in the equimolar binary mixtures. Conclusions: Combining high-throughput RNA-seq analysis and PCLS culture in Atlantic cod, we have shown that BaP and EE2 differentially regulated many genes in the AHR and ER pathways. The results show that omics data can be generated using an efficient PCLS in vitro culture system. Overall design: The seven treatment groups are DMSO (vehicle control), 10nM BaP, 1uM BaP, 10nM EE2, 1uM EE2, 10nM Mix (BaP + EE2, 10nM each) and 1uM Mix (BaP + EE2, 1uM each). Each treatment group consists of PCLS RNA samples prepared from 6-8 fish (n = 6-8 per group). A total of 47 samples were sequenced. For each sample, 50 million 75 bp paired-end reads were generated.