Illumina HiSeq 4000 sequencing; Transcriptomic profiling of clobetasol propionate-induced immunosuppression in challenged zebrafish embryos
For each sample, 10 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, the supernatant was removed and the tubes were snap frozen in liquid nitrogen.   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: Wild-type zebrafish (Danio rerio, Strain AB) broodstocks were maintained under flow-through conditions in 150 L tanks at 26 +/- 2C 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 20 fertilized eggs in the early blastula stage were placed in glass petri dishes (diameter 6 cm) filled with 8 mL of the testing solution or control medium. Embryos were incubated at 27 +/- 1C on a 14:10 h light/dark cycle. At 24 hours post fertilization (hpf), eggs were inspected while half of the test solution was renewed. Clobetasol propionate (CAS: 25122-46-7) was purchased from Sigma Aldrich (Taufkirchen, Germany). For the test solution preparation, first a 5 mM stock solution was prepared in Acetone (99.9% MS grade). This stock was diluted to 250 M in acetone before 500 L were added to a 500 mL DURAN glass bottle. To the bottle for the control solution only 500 L of pure Acetone was added. After complete evaporation of the acetone the bottle was filled up with 500 mL copper reduced tap water. Complete dissolution of precipitated clobetasol propionate was achieved by sonication for 30 minutes (Sonorex RK 100H, Bandelin, Germany).  The test solutions were freshly prepared one day prior the start of the experiment and were constantly aerated at room temperature. 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, 20 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.  Standard flagellin from Bacillus subtilis ( 10% purity), Pam3CSK4 ( 98 % purity) and Poly(I:C) HMW (high molecular weight) were purchased from InvivoGen SAS (Toulouse, France). Stock solutions (1 mg/mL for poly(I:C) and Pam3CSK4; 0.1 mg/mL for flagellin) were prepared in provided endotoxin-free water, aliquoted and stored according to the manufacturers instructions until further use. At approximately 46 hpf, the embryos were manually dechorinated and then further incubated. A mixture of the different PAMPs, was prepared by mixing 10 L of each component, resulting in final concentrations of 0.33 mg/mL for poly(I:C) and Pam3CSK4  and 0.033 mg/mL for flagellin, respectively. The device for microinjection was set up using FemtoTip II injection needles with a complementary universal capillary holder (Eppendorf, Hamburg, Germany), connected to an IM 300 microinjector (Narishige, London, United Kingdom). Volume calibration was performed according to Sive et al. (2010) using a micrometer slide with 1/100 mm scale (Bresser, Rhede, Germany). Briefly, the needles were backfilled with 10 L PAMP mix or ultra-pure water. Injections for volume calibration were made into a drop of immersion oil purred on the micrometer slide, the output pressure was constantly kept at 10 psi, while the time of injection was adjusted until an injected drop had a diameter of approximately 250 m. Assuming that the drop forms a perfect sphere, this corresponds to a volume of about 8 nL.   The procedure described below was performed for each of the five conditions in turn, treating the three replicates simultaneously. In order to anaesthetize the embryos, tricaine (Sigma Aldrich Taufkirchen, Germany) stock solution (4 mg/mL in 21 mM Tris pH 9) was added to the petri dishes to a final concentration of 200 g/mL. The immobilized embryos were transferred onto a 1 % agarose plate, which was divided into segments, one for each replicate. Excess liquid was removed so that the embryos were kept in place by surface tension. Desiccation was prevented by adding liquid, if needed. To ensure that the injections could be performed as quickly as possible, the embryos were arranged in a row in a way that their dorsal sides pointed to the same direction. The puncture was made on the dorsal side of the yolk sac and the needle was advanced through the yolk until it reached the yolk sac circulation valley on the ventral side of the embryo, where the injection was made directly into the bloodstream. After all replicates of a certain condition were processed, the embryos were rinsed back into the corresponding dish containing renewed test solution.  Total RNA was extracted using the NucleoSpin RNA/Protein kit from Macherey-Nagel (740933.250) The pooled and snap frozen larvae were suspended in RP1 buffer and were transferred to a screw-cap tubes filled with 0.3 g of Lysing Matrix D (MP Biomedicals, 6913050) ceramic beads. Tissue homogenization was performed at 5 m/s for 45 s with FastPrep-24 (MP Biomedicals, 6004500) at room temperature.  From here on RNA was extracted according to the manufacturers protocol.  RNA concentration (>100 ng/l) was measured using a Nanodrop 2000 spectrophotometer (Thermo Scientific). The samples 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 manufacturers protocol. Only samples with a RIN > 8 were used for downstream analysis. Samples were stored at -80C until they were send to the sequencing facility on dry ice.  Sequencing libraries were prepared for each sample from 100 ng/l total RNA at the sequencing facility NGS-Services for Integrative Genomics at the University of Gttingen 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). Libraries were validated using a Fragment Analyzer system (Agilent, Santa Clara, USA) before sequencing.