characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2023-05-10T00:18:42Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2023-05-10T00:18:42Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA111413247
characteristics.characteristic: External Id
characteristics.text: Fraunhofer Institute for Molecular Biology and Applied Ecology IME
characteristics.characteristic: INSDC center name
characteristics.text: 2023-05-10T00:18:42Z
characteristics.characteristic: INSDC first public
characteristics.text: 2023-05-10T00:18:42Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS13506912
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-12227:R999
characteristics.characteristic: Submitter Id
characteristics.text: 96
characteristics.unit: hour
characteristics.characteristic: age
characteristics.text: ArrayExpress
characteristics.characteristic: broker name
characteristics.text: zebrafish
characteristics.characteristic: common name
characteristics.text: Protocols: Tamoxifen (CAS: 10540-29-1) was purchased from Merck KGgA (Darmstadt, Germany). For the test solution preparation, first a high concentrated stock solution was prepared in water. From this stock, respective volumes for each testing concentration were added to a 250 ml DURAN glass bottle. Then 200 ml of Cu reduced water were added and solutions were stirred in the dark for 3 hours at room temperature. The solutions were freshly prepared one day prior the start of the experiment and stored at room temperature. They were constantly aerated and used over the course of the 96 hours exposure experiment. 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 binocular 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”. 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. 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 (>100 ng/µl) was measured using a Nanodrop 2000 spectrophotometer (Thermo Scientific). The sample’s overall RNA quality and corresponding RIN value was fluorometrically determined with a 2100 Bioanalyzer© Instrument system (Agilent, G2939BA) using the Agilent RNA 6000 Nano Kit© (Agilent, 5067-1511) according to the manufacturer’s protocol. Only samples with a RIN > 8 were used for downstream analysis. Samples were stored at -80°C until they were send to the sequencing facility on dry ice. 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 collected 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 then 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”. Adapter sequences were removed using trimmomatic (v0.39) and library’s sequence quality was assessed with FastQC (v0.11.5). Reads were checked for potential contaminations with FastQ Screen (v0.14.1) using bowtie2 (v2.3.2) 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’.
characteristics.characteristic: description
characteristics.text: larval stage
characteristics.characteristic: developmental stage
characteristics.text: wild type genotype
characteristics.characteristic: genotype
characteristics.text: mixed pool of 10 larvae
characteristics.characteristic: individual
characteristics.text: Danio rerio
characteristics.characteristic: organism
characteristics.text: Danio rerio
characteristics.characteristic: organism
characteristics.text: whole organism
characteristics.characteristic: organism part
characteristics.text: Danio rerio
characteristics.characteristic: scientific_name
characteristics.text: mixed
characteristics.characteristic: sex
characteristics.text: AB
characteristics.characteristic: strain
characteristics.text: R999
characteristics.characteristic: title