Lightcycler 480 equipment

RNA from 2D and 3D ES cultures, as well as HepG2 and MCF-7 cells, were extracted with a High Pure RNA Isolation Kit (Cat. #11828665001, Roche, Basel, Switzerland) and stored at −80 °C until use. cDNA synthesis was performed with a Transcriptor High Fidelity cDNA synthesis kit (Cat. #05091284001, Roche). RNA extraction and cDNA synthesis were both performed according to the manufacturer’s instructions. RT-qPCR was performed using SYBR-Green (SYBR Green I Master Kit, Roche), in LightCycler 480 equipment (Roche). Gene expression was determined with the comparative CT method (2^−ΔΔCT), using RPL22 and 36B4 [29 (link),31 (link)] expression as control (n = 2). The sequences of the primers used in this study are listed in Table S2. EWSR1-FLI1 translocation in ES cultures was assessed by RT-qPCR, using primers that amplify the fusion transcript between EWSR1 and FLI-1 [5 (link)]. RT-qPCR amplification products were separated by electrophoresis (2% agarose gel, Cat. #MB02703, NZYTech, Lisbon, Portugal) and amplicons were visualized using the RedSafe Nucleic Acid Staining Solution (Cat. #21141, Intron Biotechnology, Seongnam, South Korea). The same cDNAs were used to amplify 36B4 (84 bp) and examined for amplicon size comparison. A negative control (water, H₂O) for EWSR1-FLI1 was run in parallel.

To explore the immune response induced by astrocytes after 48 h of RVFV infection, total RNA was extracted according to manufacturer’s instructions (RNeasy mini kit, Qiagen, USA) and was treated for genomic DNA elimination using RNase-free DNase Set (Qiagen, USA). The quality and the concentration of the isolated total RNA from each sample were checked using the NanoDrop 2000 (Thermo Fisher Scientific, France). Immune response was then screening by a two-step RT-qPCR kit targeting 84 different genes (Human Antiviral Response, Qiagen, USA, supplementary data, Table S2) on LightCycler 480 equipment following manufacturer’s instructions (Roche, France). Cycle threshold (Ct) values of targeted genes were normalized using housekeeping genes included in the kit and fold-changes of transcripts level compared to those of uninfected condition were determined by online Qiagen Software (RT² Profiler PCR Data Analysis). If fold-change values were <1, online Qiagen Software converted value X with −1/X.