title: Illumina HiSeq 4000 sequencing; mRNA-Seq of zebrafish embryos (96hpf) exposed to different concentrations of Chlorpyrifos below acute toxicity levels against untreated control groups
design: mRNA-Seq of zebrafish embryos (96hpf) exposed to different concentrations of Chlorpyrifos below acute toxicity levels against untreated control groups
library_construction_protocol: Catalogue no. 2921-88-2. 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”. 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. Chlorpyrifos (CAS 2921-88-2, PESTANAL® analytical standard) was purchased from Merck KGgA (Darmstadt, Germany). For the test solution preparation, first a high concentrated stock solution was prepared in Acetone (99.9% MS grade). From this stock, respective volumes for each testing concentration were added to a 250 ml DURAN glass bottle and filled up with pure Acetone to a final volume of 1 ml. To the bottle for the control solution only 1ml of pure Acetone was added. The solvent was then evaporated at room temperature until no Acetone was left in the bottles. 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 in the dark. 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 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 45 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 (>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. 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). Libraries were validated using a Fragment Analyzer system (Agilent, Santa Clara, USA) before sequencing.