Illumina HiSeq 4000 sequencing; mRNA-Seq of daphnia magna juveniles exposed to different sub-lethal concentrations of Imidacloprid in a modified acute immobilisation test below against untreated control groups
mRNA-Seq of daphnia magna juveniles exposed to different sub-lethal concentrations of Imidacloprid in a modified acute immobilisation test below against untreated control groups
For each sample, all living daphnids were picked from the beaker due to reduce effects caused by dead cells. They were carefully and with a small amount of solution transferred to a screw-cap eppendorf containing a mixture of glass beads. Right after all the daphnids from each beaker were transferred, solution was removed and individuals were simultaniously euthanized in liquid nitrogen. Together with 350 µl of RP1 buffer ((NucleoSpin RNA Protein kit; Macherey-Nagel 740933.250) the samples were mechanically homogenized (See: Nucleic acid extraction protocol). RP1 buffer was freshly prepared with TCEP as reducing agent prior extraction. Origin of the daphnids is the German Federal Environmental Agency, Institut für Wasser,- Boden- und Lufthygiene. The adult daphnids were fed daily with an algal suspension (Desmodesmus subspicatus) and ArtemioFluid (JLB GmbH & Co. KG). Before test start, 3 weeks old daphnids (30 to 50) were separated from the stock population into a 1.8 L beaker containing copper-reduced tap water. On the test day, newborn D. magna were separated by sieving. In order to use only healthy daphnids, the newborn ones were transferred into the test solutions few hours after separation.   Imidacloprid (CAS 138261-41-3, ≥ 95 % purity) was purchased from Merck KGgA (Darmstadt, Germany). Test solutions were freshly prepared one day prior the start of the experiment and stored at room temperature in the dark while being constantly aerated. The glass beakers 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 100-150 daphnids were pre-picked in small glass beakers (30-50 ml) filled with ~ 20 ml of the testing solution. Based on visual observation, only healthy individuals were selected for the test. Using a plastic Pasteur pipette, 50 juvenile D. magna were transferred into the test glass beaker (250 ml, filled with 150 ml of the respective test solution (Control, Low exposure, High exposure). Daphnids were incubated at 22 +/- 1°C on a 8:16 h light/dark cycle. For a constant and even temperature distribution, all glass beakers were placed in a water filled white plastic box. Glass beakers were covered with a glass plate to avoid evaporation. The solution's pH and O2 was measured before and after the test. At 24 hours post test start, daphnids were inspected visually and dead or daphnids with abnormal behavior were noted. At 48 after test start, overall survival, abnormal behavior, morphological malformations, and physiological changes were recorded before RNA and protein extraction. Bead tubes for homogenization were filled with a mixture of sterile glass beads of 0.1 to 0.5 mm diameter. Tissue homogenization was performed at 5 m/s for 10 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 (>50 ng/µl) was measured using a Nanodrop 2000 spectrophotometer (Thermo Scientific). The sample's overall RNA quality and corresponding 28S/18S ratio was fluorometrically determined with a 2100 Bioanalyzer© Instrument system (Agilent, G2939BA) using the Agilent RNA 6000 Pico Kit© (Agilent, 5067-1511) according to the manufacturer's protocol. Only samples with a 28S to 18S ratio > 2 (reflecting good quality through intact rRNA units) were used for downstream analysis. Samples were stored at -80°C until they were sent to the sequencing facility on dry ice. Sequencing libraries were prepared for each sample from 50 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.