alias: E-MTAB-9054:R906_s
title: Illumina HiSeq 4000 sequencing; RNA-Seq of zebrafish embryos (96hpf) treated with different concentrations of 6-Propyl-2-thiouracil (6-PTU) below acute toxicity levels against untreated control groups
design: RNA-Seq of zebrafish embryos (96hpf) treated with different concentrations of 6-Propyl-2-thiouracil (6-PTU) below acute toxicity levels against untreated control groups
library_construction_protocol: For each sample, 10 4-day old zebrafish larvae were randomly picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Once individuals were picked for all samples, they were euthanized at the same time by placing the tubes simultaneously on ice for 10 minutes. Supernatant from the test solutions was carefully removed without injuring the larvae before adding 350µl of RP1 buffer (NucleoSpin RNA Protein kit; Macherey-Nagel 740933.250) for tissue homogenization. RP1 buffer was freshly prepared with TCEP as reducing agent prior extraction.  For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the “Growth protocol”. For detailed information about exposure duration and conditions please refer to the “Treatment protocol”. All samples were extracted on the 09.12.2019.  CAS: 51-52-5 Adult animal husbandry: Wild-type zebrafish (Danio rerio, Strain AB) broodstocks were maintained under flow-through conditions in 150 L tanks at 26 +/- 2°C on a 12:12 h light/dark cycle. They were fed daily with TetraMin® (Tetra Werke, Melle, Germany) main feed ad libitum and nauplii of Artemia salina. The day before test start, glass spawning-trays with artificial substrate (green glass beads stringed on stainless steel wire) were placed at the bottom of each tank. After mating and spawning in the morning hours eggs were rinsed with clear Cu-reduced water and placed into glass dishes for pre-picking.  Fish embryo incubation: For each sample 15 fertilized eggs in the early blastula stage were placed in glass petri dishes (diameter 6 cm) filled with 8 ml of the respective testing solution (Control, Low exposure, High exposure). Embryos were incubated at 27 +/- 1°C on a 14:10 h light/dark cycle. At 24 hours post fertilization (hpf), eggs were inspected visually and single coagulated eggs were recorded and removed. At 48 hpf, overall survival, hatching rates, morphological malformations and physiological changes were recorded and aged solutions were replaced by fresh, aerated test solutions. At 96 hpf again, overall survival, hatching rates, morphological malformations and physiological changes were recorded before RNA and protein extraction. 6-PTU (6-propyl-2-sulfanylidene-1H-pyrimidin-4-one, CAS-number 51-52-5,   99% purity) was purchased from Merck KGgA (Darmstadt, Germany). Test solutions were freshly prepared one day prior the start of the experiment and stored at room temperature in the dark while being constantly aerated. The glass wells for the incubation of the embryos were pre-saturated with the respective test solution overnight and renewed at the beginning of the experiment. Per sample, about 40 - 50 fertilized fish eggs were pre-picked in glass petri dishes (10 cm diameter) filled with 30 ml of the testing solution. Using an optical binokular microscope, 15 embryos of the same early blastula stage were transferred from the pre-picking plate into the test glass wells filled with 8 ml of the test solution. Embryos were then incubated as described in the “growth protocol”.  The pooled larvae in RP1 buffer were transferred to a screw-cap eppendorf filled with 0.3 g of Lysing Matrix D (MP Biomedicals, 6913050) ceramic beads. Tissue homogenization was performed at 5 m/s for 40 s with FastPrep-24© (MP Biomedicals, 6004500) at room temperature.  From here on total RNA and Protein was extracted from the tissue lysate using the NucleoSpin RNA Protein kit (Macherey-Nagel, 740933.250) according to the manufacturer's protocol.  RNA concentration was measured using a Nanodrop 2000 spectrophotometer (Thermo Scientific). The sample's overall RNA quality and corresponding RIN value was fluorometrically determined through a 2100 Bioanalyzer© Instrument system (Agilent, G2939BA) using RNApico© chips (Agilent, 5067-1513). Samples were stored at -80°C until they were send to the sequencing facility on dry ice. Sequencing libraries were prepared for each sample from 100 ng/µl total RNA at the sequencing facility “NGS-Services for Integrative Genomics” at the University of Göttingen in Germany. According to their standard workflow, cDNA libraries were prepared from protein coding mRNA that were purified through PolyA selection using the TruSeq RNA Library Prep Kit v2 (Illumina).
identifiers.namespace: Fraunhofer Institute for Molecular Biology and Applied Ecology, Applied Ecology and Bioresources Division, Schmallenberg, Germany  Institute of Ecology, Evolution and Diversity, Goethe University Frankfurt, Frankfurt am Main, Germany
identifiers.id: E-MTAB-9054:R906_s
attributes.tag: Experimental Factor: compound
attributes.value: 6-propyl-2-thiouracil
attributes.tag: Experimental Factor: dose
attributes.value: 0.1