Tri reagent ls

The porcine embryo kidney cell line’s total RNA was used to normalise the amount of total RNA in each sample during reverse transcription and qPCR. To isolate the total RNA of the porcine embryo kidney (PEK) cell line, 750 μL of the TRI reagent LS (Sigma-Aldrich, St. Louis, MO, USA) was added to the one-day PEK cells’ monolayer that was growing on the 25 cm² cell culture flask (Corning, New York, NY, USA), with the cell supernatant being discarded before the procedure. The TRI reagent LS was then used to lyse the PEK cells, and the obtained mixture was used to proceed with the RNA isolation protocol described in Section 2.2.
The purified RNA was dissolved in 50 μL of water, the RNA concentration was measured by using NanoDrop One^C (ThermoFischer Scientific, Madison, WI, USA), and the RNA was subsequently aliquoted to 250 ng/μL and stored at −70 °C.

To extract SARS-CoV-2 RNA, 200 µL of food rinsing fluid from each food sample was mixed with an equal volume of TRI Reagent LS (ThermoFisher, Waltham, MA, USA). Viral RNA was extracted and purified using Direct-zol^TM RNA microPrep kit (Zymo Research, Irvine, CA, USA). A NanoDrop 2000 spectrophotometer (ThermoFisher, Waltham, MA, USA) was used to measure concentrations and purity of the extracted viral RNA using reading absorbances at 260 nm and 280 nm. To determine RNA genome copy number, 10 µL qRT-PCR reactions were prepared using the iTaq Universal Probe One-Step Kit (BioRad, Hercules, CA, USA), mixed with primers and probes specific for SARS-CoV-2 N protein, and then amplified on a ViiA 7 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) [33 ]. The standard setting used for qRT-PCR is as follows: 1 cycle of 10 min at 50 °C followed by 2 min at 95 °C; and 45 cycles of 3 s at 95 °C and 30 s at 55 °C. Final results were reported as genome copy number per mL of food rinsing fluid, to allow for direct comparison to infectious viral titer measured in PFU/mL.

RNA was extracted from EVs and filament samples using standard methods for TRI-reagen LS (Sigma) and TRI-reagent (Sigma), respectively, with a few modifications. Extracted RNA was treated with DNase I (ThermoFisher) as per manufacturer’s instructions and re-extracted with TRI-reagent LS. Coprecipitant GlycoBlue^TM (ThermoFisher) was used for EV RNA samples for the first extraction and for all samples in the re-extraction. RNA quality was controlled with Bioanalyzer^TM RNA 6000 Nano (Agilent, Santa Clara, CA, USA) assay using the eukaryote setting. Libraries for sequencing were generated with NEBNext^® Ultra™ II Directional RNA Library Prep Kit for Illumina together with NEBNext^® Poly(A) mRNA Magnetic Isolation Module according to the manufacturer’s instructions (NEB, Frankfurt am Main, Germany). Libraries were quality controlled with High Sensitivity DNA Kit on Bioanalyzer (Agilent) and quantified on Qubit 2.0 Fluorometer (ThermoFisher) with ds HS Assay Kit. Sequencing was performed in the Genomics Service Unit of LMU Biocenter, on Illumina MiSeq with v3 chemistry with 2 × 150 bp paired-end reads (Illumina, San Diego, CA, USA).