Rnase one

To determine the contribution of free PV RNA in stool suspensions to the overall RNA content measured by RT-qPCR, 130 μL PV2-positive stool suspension (Sabin-like 2, 10^5.88 CCID₅₀ /100 μL) and 100 μL virus reference isolate (Sabin 1, 10^6.2 CCID₅₀) was incubated with 250 units of Benzonase (MilliporeSigma, St. Louis, MO) and 20 units RNAse ONE (Promega, Madison, WI) for 2 h at 37°C, each. Samples were spiked with a PV VP1 RNA transcript derived from a WPV3 virus at a concentration of 10⁶ copies·μL^-1 as a positive control for the nuclease activity. The same specimens (baseline samples) were processed simultaneously without nucleases. Following the RNA extraction, 5 μL template RNA were assayed by RT-qPCR (ITD kit) with PV VP1 RNA transcripts (Sabin 1, Sabin 2, or WPV3).

RNA was extracted from 14-day-old plants as described previously^{63 (link)}. The first strand of cDNA was prepared with the PrimeScript RT Reagent Kit (TAKARA, RR037A). Quantitative PCR was performed on an ABI 7500 fast Real time PCR instrument with KAPA SYBR FAST Universal reaction regent, and the results were quantified by reference to a standard curve for each primer pair with at least two repeats. For assessment of noncoding RNA expression, sequence-specific primers were used for reverse transcription. For RNase-treated RT-qPCR, 10 µg of total RNA was digested with RNase III (NEB, M0245S) or RNase one (Promega, M4261) according to the instruction manual. After chloroform purification, 1 µg of RNA was treated with DNase I to remove genomic DNA, and the RNA was then subjected to RT-PCR. Oligonucleotides used in this study are indicated in Data set 2.

In order to obtain the complete genome sequences of samples identified as CoV-positive in our earlier studies (166 positives including 140 gamma- and 26 deltacoronaviruses), selected samples with the highest viral load confirmed by PCR were subjected to Next Generation Sequencing (NGS) using the MiSeq Personal Sequencer platform (Illumina, USA) offered by the Department of Omics Analysis of PIWet-PIB or the commercial service Genomed SA. (Warsaw, Poland). A total of 27 coronavirus-positive field specimens were subjected to NGS attempts. Briefly, samples (cloacal swabs) were treated with TURBO DNase (Life Technologies, USA) and RNase One (Promega, USA) to remove DNA and extracapsid RNA. The isolation of viral RNA from such treated samples was carried out and then retrotranscribed into DNA using a Superscript IV First-Strand cDNA Synthesis Kit (Invitrogen, USA) and the second strand was synthesized with the addition of Klenow polymerase (New England Biolabs, USA). A 300 bp long paired-end DNA library was prepared using a Nextera XT sample preparation kit (Illumina Inc) and sequencing was performed using a MiSeq Reagent kit v3 (Illumina Inc).

For all ISVs, fluid supernatant from cultures of infected C6/36 cells were used for RNA extraction and sequencing. Supernatants were harvested and clarified by low speed centrifugation (2,000 × g, 10 minutes at 4°C) once CPE was advanced. One milliliter of clarified supernatant from each virus was treated with a cocktail of DNases (14 U Turbo DNase [Ambion, Austin, TX], 20 U Benzonase [EMD Millipore, Billerica, MA], and 20 U RNase One [Promega, Madison, WI]) for 1 hour at 37°C. Viral RNA was then extracted using Trizol and resuspended in 50 μL RNase/DNase and protease-free water (Ambion, Austin, TX).

The CAGE libraries from the purified RNA samples were prepared as described previously^{22 (link)}. In brief, first strand cDNA synthesis was performed by reverse transcription with SuperScriptIII (Thermo Fisher Scientific), the diols in the cap structure of the ribose sugar and at the 3′ end of the RNA were oxidized with sodium periodate, and then biotinylated with biotin (long arm) hydrazide (Vector Laboratories, Burlingame, CA, USA). Single-stranded RNA portions were digested with RNase ONE (Promega, Fitchburg, Wisconsin, USA), and then biotinylated RNA/cDNA was captured on the surface of Dynabeads M-270 streptavidin (Thermo Fisher Scientific). The cDNA was released by heat denaturation and purified by RNase ONE/H digestion followed by AMPure XP (BioRad, Hercules,CA, USA) purification. The purified single-stranded cDNA was subjected to adaptor ligation at both ends, and the double-stranded cDNA library was created using DeepVent (exo-) DNA polymerase. The generated CAGE cDNA libraries were sequenced by an Illumina HiSeq 2500 sequencer (Illumina, San Diego, CA, USA).

Sediment in the microcosms was sampled at four different time points (Supplementary Figure S1). First samples were taken right at the onset of the experiment and equaled the initial sediment; all other samples were collected after 9, 21, and 43 days of incubation, which were preceded by 7 days of pre-incubation. Sediment samples were frozen immediately in liquid N₂ and stored at -60°C until further processing. RNA and DNA were extracted from the same samples using the RNA PowerSoil^® Total RNA Isolation Kit in combination with the RNA PowerSoil^® DNA Elution Accessory Kit (Mo Bio Laboratories Inc., Carlsbad, CA, United States). Residual DNA in the RNA was removed with the TURBO DNA-free Kit (Ambion, Thermo Fisher Scientific, Darmstadt, Germany). Transcription of RNA into cDNA was performed using SuperScriptIII (Life Technologies, Darmstadt, Germany). To remove traces of remaining RNA, extracted DNA was treated with RNase ONE (Promega, Mannheim, Germany). RNA and DNA were quantified using Ribo- and PicoGreen (Life Technologies), respectively.