R3529
Protocols: Tamoxifen (CAS 54965-24-1) was purchased from Merck KGgA (Darmstadt, Germany). The test solution was prepared by first preparing a highly concentrated stock solution in water, stirring for approximately 30 minutes. The test solutions were prepared from these stock solutions immediately before the start of the test to avoid any degradation of the substance. All tamoxifen test solutions were prepared in copper reduced tap water. They were constantly aerated and used over the course of the 96 hours exposure experiment. The glass wells for incubation of the embryos were pre-saturated with the respective test solution overnight and renewed at the beginning of the experiment. Using a binocular optical microscope, 15 embryos of the same early blastula stage were transferred from the pre-picking plate to the test glass wells filled with 8 ml of test solution. The embryos were then incubated as described in the “growth protocol”. 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 Artemia salina nauplii. One day before the start of the test, glass spawning trays containing artificial substrate (green glass beads strung 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 (0, 0.1265, 0.4, 1.265, 4, 12.65, 40 µg/L). 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 extraction. Total RNA and Protein were extracted from the tissue lysate using the NucleoSpin RNA Protein kit from Macherey-Nagel following the manufacturer's protocol. The RNA concentration was measured using a Nanodrop 2000 spectrophotometer from Thermo Scientific, with a minimum concentration of 100 ng/µl required. The overall RNA quality and RIN value were determined using the Agilent 2100 Bioanalyzer system and the Agilent RNA 6000 Nano Kit, respectively. Only samples with a RIN value greater than 8 were used for downstream analysis. The samples were stored at -80°C until they were sent to the sequencing facility on dry ice. For each sample, 10 zebrafish larvae were collected and transferred to a low binding Eppendorf tube. The larvae were euthanized simultaneously by placing the tubes on ice for 10 minutes. The supernatant was carefully removed without injuring the larvae, and RP1 buffer from the NucleoSpin RNA Protein kit (Macherey-Nagel 740933.250) was added for tissue homogenization. The RP1 buffer was prepared with TCEP as a reducing agent. Detailed information about embryonic incubation conditions, husbandry of adult broodstocks, exposure duration, and conditions can be found in the \"Growth protocol\" and \"Treatment protocol\" respectively. Adapter sequences were removed using trimmomatic (v0.39) and library's sequence quality was assessed with FastQC (v0.12.0). Reads were checked for potential contaminations with FastQ Screen (v0.15.3) using bowtie2 (v2.5.3) against the following genomes: Daphnia magna (ftp://ftp.ensemblgenomes.org/pub/metazoa/release-48/fasta/daphnia_magna/dna/Daphnia_magna.daphmag2.4.dna.toplevel.fa.gz), Homo sapiens, Mus musculus, Drosophila melanogaster, Saccharomyces cerevisiae, Eschericia coli as well as custom build human rRNA database. With the exception of D. magna, pre-built Bowtie2 indices for those genomes were directly downloaded from Babraham Bioinformatics with the built-in function 'fastq_screen --get_genomes'.
strain
wild type
individual
mixed pool of 10 larvae
organism
Danio rerio
organism
Danio rerio
collection date
not collected
sex
mixed
scientific_name
Danio rerio
common name
zebrafish
organism part
whole organism
developmental stage
larval day 4
genotype
wild type genotype
isolate
not applicable
age
96 (hpf)
geographic location (country and/or sea)
not collected