Illumina HiSeq 4000 sequencing; mRNA-Seq of Lemna minor exposed to different concentrations of the pharmaceutical atorvastatin against untreated control groups
mRNA-Seq of Lemna minor exposed to different concentrations of the pharmaceutical atorvastatin against untreated control groups
For each sample, 25 mg of plant tissue were randomly collected from the test vessel into a tube. After adding the lysis matrix (¼ Ceramic Sphere, MP Biomedicals), the lysis was performed according to the manufacturer's protocol of the RapidPURE RNA Plant Kit (MP Biomedicals Illkirch, France). Homogenization was conducted at 5 m/s for 60 s with FastPrep-24© 5G (MP Biomedicals Illkirch, France) at room temperature. One week before test start, a pre-culture of Lemna minor was auxenically prepared from a stock culture in Steinberg medium according to OECD TG 221. Plants were kept in a growth chamber at 24±2°C and under continuous light (85-135 µE∙m-2∙s-1). Atorvastatin-calcium was purchased from abcr (Karlsruhe, Germany). Test solutions of EC5 and EC20 (% inhibition of growth (e.g. 50 %) is determined and expressed as the ECx e.g. EC50) determined in preliminary experiments according to OECD TG 221, were prepared with Steinberg medium at a pH 5.5±0.2. 150 mL of each test solution were transferred into three test vessels (replicates) and analogously 150 mL of pure Steinberg medium were used as control samples. Four healthy plants with three fronds each were transferred from the pre-culture to the test vessels. Incubation was conducted according to the conditions described in “Growth protocol” under random arrangement for 3 days. Total RNA and protein were extracted according to the RapidPURE Plant Kit (MP Biomedicals Illkirch, France). RNA quantification (RNA>100 ng/µL) was performed using Nanodrop 2000 spectrophotometer (Thermo Scientific), and quality control (RIN>7.0) was conducted using the 2100 Bioanalyzer (Agilent Technologies). The samples were stored at -80°C until mRNA sequencing.  cDNA libraries were prepared from mRNA that was 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.