Dnase 1

The 2 µL of EVs was incubated at room temperature for 15 min with 1 U of DNase I (Zymo Research, Irvine, CA, USA) and 1× DNA Digestion Buffer. The plasmid DNA was isolated from the EVs using Qiamp Miniprep kits and quantified by qPCR. The plasmid DNA was also extracted from nontreated EVs as a negative control.

To identify the role of nucleic acids or proteins in the transfer of EV-mediated TIG-R, TIG-R EVs (5 μg) were treated as previously described [24 (link),25 (link)] with minor modifications. Briefly, 2.5 μL of DNase I (1 U∙µL⁻¹, Zymo Research, Irvine, CA, USA), Sau3AI (4000 U∙mL⁻¹, New England Biolabs, Ipswich, MA, USA), or proteinase K (20 mg∙mL⁻¹, Thermo Fisher Scientific, Waltham, MA, USA) were treated with TIG-R EVs (5 μg) for 2 h at 37 °C according to the manufacturers’ instruction. The resulting EV fractions were characterized by 1% agarose gel electrophoresis, 12% Mini-PROTEAN® TGX^TM Stain-Free gel (Bio-Rad, Hercules, CA, USA) electrophoresis, and imaging analysis. The resulting samples were used to analyze the transference of TIG-R to the TIG-S AB strain.

DNA was extracted from ~300 mg reactor sample using the NucleoSpin Soil Kit (Machery-Nagel, Düren, Germany) following the instructions of the manufacturer. Extracted DNA was stored at −20°C.
RNA was extracted from 0.5 g of reactor sample. Samples were frozen in liquid N₂ and homogenized using an dismembrator instrument (Sartorius AG, Göttingen, Germany) with 2000 rpm for 5 min. Samples were transferred into 2.5-ml Isoyl-RNA Lysis reagent (5 Prime GmbH, Hilden, Germany) and RNA was extracted applying the Direct-zol RNA Kit (Zymo Research, Freiburg, Germany). The protocol for RNA purification included on-column DNase I treatment (5 μl, 6 U/μl, Zymo Research, Freiburg, Germany) according to manufacturer instruction. Extracted RNA was stored at −80°C until further preparation.

Total RNA from CMs transfected with siSphk1 or siNC and treated with AngII or a control buffer (Ctrl) was isolated using a Direct-zol RNA Miniprep kit and treated with DNase I (Zymo Research, Irvine, CA, USA). Reverse transcription was performed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). Real-time PCR reactions were performed using commercially available TaqMan assays, Taqman Gene Expression Master Mix and the 7900HT instrument (Applied Biosystems, Foster City, CA, USA). TATA box binding protein (Tbp) was used as a housekeeping gene for all performed real-time PCR reactions. Ct values were calculated using RQ Manager (Applied Biosystems, Foster City, CA, USA).

Eighteen stool samples (10–15 gr) were collected in 2020 in sterile bags from a small-sized farm situated in Salto City, where the highest seroprevalence of HEV had been reported in a previous study [16 (link)]. Samples were kept on dry ice and sent to the laboratory where they were processed.
Pig fecal samples were resuspended at 10% w/v in sterile phosphate buffer saline (PBS) and vigorously vortexed. After centrifugation for 30 min, 8000× g at 4 °C, the supernatant was filtered to 0.22 μM. Total RNA was extracted with a Quick-RNA™ Miniprep Kit and treated with DNase I (Zymo Research Corp., Tustin, CA, USA) according to the manufacturer’s instructions.
For the detection of HEV, RNA was subjected to RT-nPCR targeting a 330-bp region within ORF2 as previously reported [17 (link)]. PCR products were gel-visualized under UV light and amplicons of the expected size were sequenced in both directions by Macrogen Inc. (Seoul, Republic of Korea).
The HEV-positive samples were filtered (0.45 μM) and viral particles were concentrated in a 30% sucrose gradient and ultra-centrifuged for 2:30 h at 100,000× g, 4 °C in a Sorvall™ WX+ Ultracentrifuge (Thermo Scientific, Waltham, MA, USA). The pellet was resuspended in 200 µL of PBS 1× and RNA was then extracted as mentioned above.