mRNA-Seq of Lemna minor exposed to different concentrations of sucralose against untreated control groups
For each sample, 30 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). Sucralose (CAS 56038-13-2, purity 98%) was purchased from abcr. Test solutions of 10 µg/L and 100 mg/L were prepared with Steinberg medium. 150 mL of each test solution were transferred to three test vessels (replicates) and analogously 150 mL of Steinberg medium were used as control samples. 4 plant with 3 fronds each were transferred to the test vessels. Incubation was carried out in accordance with OECD 221 at 24±2 °C and continous light. 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 -20°C until mRNA sequencing.  Poly(A)-enriched, strand-specific mRNA libraries were generated by the sequencing provider (Novogene) using a standard Illumina-compatible workflow comprising poly(A) selection, RNA fragmentation, first- and second-strand cDNA synthesis, adapter ligation, PCR amplification and size selection, and the resulting libraries were sequenced as 150 bp paired-end reads on an Illumina NovaSeq 6000 platform at a target depth of approximately 30 million read pairs per sample.