mRNA-Seq of Danio rerio exposed to different concentrations of sucralose against untreated control groups
For each sample, 10 zebrafish embryos 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 45s 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 copper reduced tap water. 7.5 mL of each test solution were transferred to three test vessels (replicates) and analogously 7.5 mL of water were used as control samples. 15 freshly fertilized eggs were transferred to the test vessels. Incubation was carried out in accordance with OECD 236 at 27±1 °C and a light/dark cycle of 14:10 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 = 50 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 8 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.