Name: Sample 2_p
Instrument: Illumina NovaSeq 6000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: PolyA
Layout: PAIRED
Construction protocol: At 72 hpf, for each sample, all 15 zebrafish embryos were picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Excessive liquid was removed and tubes were placed on ice to euthanize the embryos. 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”. Adult animal husbandry: All experiments were performed using zebrafish (Danio rerio) embryos of the wild-type strain AB. Adult fish were originally obtained from West Aquarium GmbH, Germany, and were kept in 150 l tanks under flow-through conditions at 26 ± 2 °C and a 12:12 light/dark cycle at the Fraunhofer Institute for molecular biology and applied ecology (IME), Schmallenberg, Germany. TetraMin® (Tetra Werke, Germany) was used as daily main feed, regularly supplemented with nauplii of Artemia salina. Fish were constantly bred for several generations without interbreeding between tanks, making the embryos deriving from different spawning groups (tanks) independent biological replicates. For egg collection, glass spawning trays equipped with whirled cords of green glass pearls to stimulate spawning and covered with wired mesh to prevent cannibalism were put into the tanks the day before tests were started (OECD, 2013). The morning after, eggs were collected from the trays using a sieve, rinsed with pre-tempered system water to remove remaining feces, gathered in glass jars filled with pre-tempered aerated system water and stored at a controlled temperature of 26 ± 1 °C until further use. Fish embryo incubation: Embryos were incubated at a controlled temperature of 26 ± 1°C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. For details on the treatment see “treatment protocol” Clobetasol propionate (21-Chlor-9-fluor-11β,17-dihydroxy-16β-methylpregna-1,4-dien-3,20-dion-17-propionat, purity ≥ 98 %, CAS no. 25122-46-7) and Imiquimod (1-(2-methylpropyl)-1H-imidazo[4,5-c]quinoline-4-amine, purity ≥ 95 %, CAS no. 9901-02-6) were purchased from BIOMOL GmbH, Hamburg, Germany. For IMQ, a stock solution in acetone with a nominal concentration of 0.1 mg/ml was prepared. For CP, a stock solution in dimethyl sulfoxide (DMSO) with a nominal concentration of 2.5 mM was prepared. To obtain a test solution with a nominal concentration of 4666 nM IMQ, 2242.5 µl of the IMQ stock solution was added to a glass bottle. To equalize the amount of added acetone amongst all solutions, equal volume of pure acetone without IMQ was added to glass bottles for all remaining test solutions. For all test solutions containing 250 nM CP, 20 µl of the CP stock solution was added to the glass bottles after complete evaporation of the acetone and filled with copper reduced tap water (in the following referred to as cu-red water) to a final volume of 200 ml. Test solutions without CP were prepared alike adding 20 µl DMSO without CP. All solutions were sonicated for 30 min at room temperature (Sonorex RK 100H, Bandelin, Germany). Final solutions were thus cu-red water with 0.01 % DMSO, 4666 nM IMQ in cu-red water with 0.01 % DMSO, 250 nM CP in cu-red water with 0.01 % DMSO and 4666 nM IMQ plus 250 nM CP in cu-red water with 0.01 % DMSO. Due to a mandatory dilution during the methodological procedure a nominal working concentration of 4000 nM was achieved for IMQ in these solutions. The test solutions were freshly prepared one day prior the start of the experiment and stored at room temperature in the dark. They were constantly aerated and used over the course of the 72 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. In order to minimize the differences between replicates and conditions in exposure durations, the selection of embryos was performed in two steps: at 2 hpf, approx. 50 embryos from spawning batches with a minimum fertilization rate ≥ 90 % were transferred to previously overnight saturated glass petri dishes (6 cm diameter) refilled with 7 ml of the corresponding test solutions for each replicate and condition. Embryos of the control and the IMQ condition were thus transferred to medium without any compound, embryos of the CP and combined CP+IMQ condition were transferred to medium supplemented with 250 nM CP. Once in solution, 15 healthy and fertilized embryos, indicated by a well-shaped blastodisc between the 128-cell and 1k-cell-stage of development were selected per replicate and condition for experiments. Embryos were incubated at a controlled temperature of 26 ± 1°C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. Embryos were inspected microscopically on a daily basis and at the end of the experiment using an OZL 451 stereomicroscope (KERN & Sohn, Germany) in order to monitor the occurrence of indicators of lethality as defined by the OECD FET test guideline, namely coagulation, lack of somite formation, lack of a heartbeat, non-detachment of the tail (OECD, 2013) as well as further indicators of malformations such as scoliosis, formation of edema or lack of pigmentation. At 47 hpf, embryos were manually dechorionated using two pointy forceps. At 48 hpf, the immune-challenge was started by transferring the embryos of the IMQ condition and the combined CP+IMQ condition to the respective test solution supplemented with IMQ. For this and in order to equalized the subsequent dilution of media between replicates and conditions, embryos were gathered in 1 ml of the aged medium and transferred with this volume into newly saturated glass dishes, filled with 6 ml of the corresponding fresh medium. Due to this dilution, a nominal working concentration of 4000 nM IMQ was achieved for the immune-challenge. Embryos of the control condition and the CP condition were handled alike but without changing the type of test solution. RNA was extracted using a NucleoSpin RNA/Protein kit (Macherey-Nagel, Düren, Germany). Briefly, frozen embryos were transferred to screw cap tubes filled with 350 µl kit specific lysis buffer (RP1 buffer) and Lysing Matrix D ceramic beads (MP Biomedicals, Irvine, USA) and homogenized using a FastPrep-24 (MP Biomedicals, Irvine, USA) device at 5 m/s for 1 min. RP1 buffer was freshly prepared with TCEP as reducing agent prior extraction. Further steps were performed following the manufacturer's extraction protocol. As quality measures, purity and concentration of extracted RNA were determined using a NanoDrop 2000 device (Thermo Scientific, Waltham, USA) and RNA integrity numbers (RIN) were determined using a Bioanalyzer 2100 device (Agilent, Santa Clara, USA) with samples prepared using a RNA Pico 6000 kit (Agilent, Santa Clara, USA) following the manufacturer's instructions. Only samples with a RIN > 9 were used for downstream analysis. Samples were stored at -80°C until they were send to the sequencing facility on dry ice. High quality RNA samples with RIN > 9 were selected for RNA-seq library preparation and sequencing, performed by Eurofins Genomics Germany GmbH, Ebersberg, Germany. Briefly, Poly-A containing protein coding mRNA molecules were purified from total RNA, randomly fragmentated and subjected to random-primed strand-specific cDNA library preparation using the TruSeq RNA library Prep Kit v2 (Illumina) following the manufacturer's instructions.