mRNA-Seq of Daphnia magna exposed to different concentrations of sucralose against untreated control groups
For each sample, 10 daphnia were randomly transferred from the test vessel into a tube. After adding the lysis buffer (NucleoSpin® RNA/Protein, Macherey-Nagel GmbH & Co. KG, Düren, Germany), lysis was performed according to the manufacturer's protocol for the NucleoSpin® RNA/Protein extraction kit. Homogenization was conducted at 5 m/s for 10 s with FastPrep-24 (M.P. Biomedicals, Irvine, CA, USA) at room temperature. Sucralose (CAS 56038-13-2, purity 98%) was purchased from abcr. Test solutions of 10 µg/L and 100 mg/L were prepared with Elendt-M4 medium. 40 mL of each test solution were transferred to three test vessels (replicates) and analogously 40 mL of pure Elendt-M4 medium were used as control samples. 20 juvenile daphnia were transferred to the test vessels. Incubation was carried out in accordance with OECD 202 in complete darkness over a period of 48 hours. Total RNA and protein were extracted according to the NucleoSpin® RNA/Protein extraction kit (NucleoSpin® RNA/Protein, Macherey-Nagel GmbH & Co. KG, Düren, Germany). RNA quantification (RNA > 10 ng/µl) was performed using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA), and quality control was conducted using the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The samples were stored at -80 °C until mRNA sequencing. Only samples showing RIN values of at least 5 were proceeded to library preparation. Poly(A)-enriched, strand-specific mRNA libraries were then 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.