title: Estrogen and xenoestrogen modulated transcriptome in the pituitary of juvenile female Atlantic cod (Gadus morhua)
design: Estrogen and xenoestrogen modulated transcriptome in the pituitary of juvenile female Atlantic cod (Gadus morhua)
library_name: P-15_FID57_p
library_construction_protocol: Atlantic cod (G. morhua) were purchased from Havbruksstasjonen in Tromsø AS (Nofima, Tromsø, Norway) and kept in 500 L tanks in 10°C, 34 ppt seawater with a 12 h light/l2 h dark cycle at the Industrial and Aquatic Laboratory (Bergen, Norway). The fish, approximately 1.5 years old, body weight (bw) 332  69g (mean  standard deviation/) were fed with a commercial diet (Amber Neptune, Skretting, Stavanger, Norway) until the start of the treatment.  The fish were not fed during exposure. In this experiment (Experiment 1), the exposure groups consisted of vehicle control, low, medium or high doses of each of EE2, BPA and MXC. Each of the compounds EE2, BPA and MXC were dissolved in vehicle (1% DMSO, 25% kolliphor in PBS) and injected at low, medium or high doses of EE2: 10, 50 or 250 nanomole (nmol)/kg body weight (bw); BPA: 8, or 40 micromole (μmol)/kg bw; and MXC: 8 or 40 μmol/kg bw. Sampling was performed 3 days after injection by killing the fish with a blow to the head. Biometric data, body weight, liver weight, gonadal weight, body length, and sex were registered. After dissection of each fish, a small piece pf liver tissue was sampled and the whole pituitary gland was dissected and snap-frozen individually in liquid nitrogen and stored at -80 °C until RNA extraction.  Total RNA extraction from each frozen pituitary was performed using TRI Reagent (Sigma) according to the manufacturer's protocols. The concentration and quality of total RNA was assessed using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA), and the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA).  For experiment 1, a total of 40 pituitary samples (n = 4-6 per group) were analyzed. RNA sequencing was performed at the Genomics Core Facility at the University of Bergen, as previously described (Yadetie et al., 2018). RNA samples from individual pituitary samples (0.4 μg total RNA per sample) were sequenced according to Illumina TruSeq® Stranded mRNA Sample Preparation Guide on Illumina HiSeq 4000 (Illumina, Inc., San Diego, CA, USA).