Extraction and Purification of dsRNA from Stool

The extraction of dsRNA was performed at the MRC-DPRU and involved a method previously described by Nyaga et al. (2018)^{53 (link)}. Briefly, a 100 mg stool sample was added to 200 µL freshly made phosphate buffered saline (PBS) (Sigma-Aldrich, Germany). A 900 µL volume of TRI-REAGENT-LS (Molecular Research Center, Inc, Cincinnati, OH, USA) was added to the suspended stool sample to homogenize as well as lyse the cells and cell components. A volume of 300 µL chloroform (Sigma-Aldrich, Germany) was then added and centrifugation (13,000 rpm for 20 min at 4 °C) was done in a temperature-controlled microcentrifuge (Eppendorf centrifuge 5427R, Hamburg, Germany). The supernatant containing the total RNA was precipitated by addition of 700 µL isopropanol (Sigma-Aldrich, Germany) and by centrifugation at 16,000×g for 30 min at room temperature. The resulting pellet was re-dissolved by addition of 90 µL of ddH20 (Merck KGaA, Germany). A concentration of 8 M LiCl₂ (Sigma, St. Louis, MO, USA) was used to remove ssRNA through precipitation for 16 h after which further centrifugation was done for 30 min at 16,000×g. The extracted dsRNA was purified by utilizing the MinElute gel extraction kit (Qiagen, Hilden, Germany) and the integrity and enrichment of the dsRNA was verified via agarose gel electrophoresis.