Rnase one ribonuclease

The dsRNA protection assay was performed as described previously (Okano et al., 2014 (link)) with a few modifications. In brief, extracted total RNA (5 μg) was treated with DNase I and incubated with the indicated concentrations of RNase One Ribonuclease (Promega) for 1 h at 37°C. RNase-treated RNA was used as a template for reverse transcription using random primers. The resulting cDNA was used in a qPCR with oligos found in Supplemental Table 2. The data were further analyzed in Microsoft Excel, and relative steady-state levels were calculated by comparison with the level of ACT2 in samples without RNase treatment. For dsRNA-IP-qPCR, nuclei from 3 g of 12-day-old seedlings were isolated with Honda buffer (0.44 M sucrose, 1.25% Ficoll, 2.5% Dextran T40, 20 mM Tris-HCl [pH 7.4], 10 mM MgCl₂, and 0.5% Triton-X). After addition of lysis buffer (0.3 M NaCl, 20 mM Tris-HCl [pH 7.4], 5 mM MgCl₂, 5 mM BME, 0.5% Tween, and RNase and protease inhibitors) and centrifugation, the supernatant was mixed with a dsRNA-specific antibody (J2, Jena Biosciences), and IP was performed as described previously (Gao et al., 2020 (link)). The isolated RNA was used as a template in a room temperature reaction with Superscript IV (Invitrogen) and gene-specific oligos (Supplemental Table 2) and then used in qPCR reactions. IP samples were compared to input samples for further analysis.

Of the 200 μL of propagated viruses collected from the apical surface of HBEC3-ALI cultures 7 days after inoculation with 8 log₁₀ RNA copies/well of RV-C9 and -C53, 6 μL was used for RNase treatment. A total 100 μL of reaction mixture containing 10 μL of 10× reaction buffer, 1 μL of RNase ONE™ Ribonuclease (10 U/μL; Promega, Madison, WI, USA), 83 μL of water, and 6 μL of sample was incubated for 30 min at 37 °C. To prepare the untreated samples, 1 μL of water was added to the reaction mixture instead of RNase ONE™ Ribonuclease.

The supernatant from the pooled sample preparation was filtered through a Millex-HPF HV 0.45 μm filter (Merck Millipore) by use of a 2-ml syringe to remove bacterial and host cells. In total 180 μl of the filtrate was treated with 20 μl Turbo DNase (Ambion) and 2 μl RNase ONE Ribonuclease (Promega) at 37°C for 30 min to degrade nucleic acids not protected in viral capsids. Nuclease digestion was stopped by adding three volumes (600 μl) of TRI Reagent LS (Sigma-Aldrich) to each sample and mixed for 5 min. An equal amount of ethanol (99,5%) was added and mixed thoroughly before the mixture was transferred into a Zymo-Spin column placed in a collection tube (Direct-zol RNA MiniPrep Kit, Zymo Research). The columns were centrifuged at 16000 x g for 30 sec. after which they were washed two times with 400 μl of PreWash solution each time followed by 30 sec. of centrifugation. Then 700 μl of RNA Wash Buffer was added to the columns followed by centrifugation for 2 min before they were transferred to RNase free Eppendorf tubes. RNA was eluted from the column matrix by adding 30 μl of DNase/RNase free water and centrifugation at 16,000 x g for 30 sec.

The spleen and kidneys of an adult female Rungwe brush-furred rat, snap-trapped in July 2011 in an old banana plantation starting to be overgrown by Afromontane forest on Mount Mabu, Mozambique (coordinates: 16.3086S, 36.4245E), were collected and stored in RNAlater (QIAGEN Benelux, Venlo, The Netherlands). RNA was extracted from one of the kidneys according to the viral enrichment protocol S3, described in [35 (link)], using 25 U RNase ONE Ribonuclease (Promega Benelux, Leiden, The Netherlands) and 30 U Benzonase Nuclease (Sigma-Aldrich, St. Louis, MS, USA) for RNA digestion (90 min at 37 °C). Elution was performed twice by collecting and loading the same eluate on the column. Subsequently, the sample was quantitated using the RNA Quantifluor System (Promega) and an Agilent RNA 6000 Pico chip, loaded on a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). Viral RNA was subjected to pre-amplification, using the Ovation RNA-Seq System V2 (NuGEN Technologies, San Carlos, CA, USA) for cDNA generation. The sequencing library was constructed with the Ovation UltraLow Library System V2 (NuGEN Technologies) and paired-end sequencing was performed on an Illumina NextSeq 500 (Illumina, Hayward, CA, USA).

Total RNA was extracted using NucleoSpin^® RNA (Macherey-Nagel) and 3 μg of the total RNA were reverse-transcribed using superscript III (Thermo Fisher Scientific Inc.). Cap structure of the RNA was biotinylated, followed by treatment of RNase ONE ribonuclease (Promega Corporation, Madison, WI, USA), RNA-cDNA hybrids were captured using streptavidin-coated magnetic beads (Thermo Fisher Scientific Inc.), and only single-stranded cDNAs were released from the beads. The released cDNAs were ligated to linkers and the second strand were synthesized using Deep Vent (exo-) DNA polymerase (New England BioLabs, Ipswich, MA, USA). The CAGE libraries were sequenced using single-end reads of 50 bp on the Illumina HiSeq 2500 (Illumina). The extracted CAGE tags were then mapped to the human hg19 genome by STAR. The tags per million (TPM) were calculated for each FANTOM5 TSS peak and regions extended ±250 bp from each differentially methylated CpG. Gene expression levels of each gene were computed as the sum of multiple TSS peaks associated with a single gene. The CAGE analysis was performed in three biological replicates.

TotalRNA was isolated from cells and exosomes using miRNeasy Mini Kit (Qiagen; Germany). Previously, free-circulating RNA was removed by resuspending the exosome pellet in 100 μl PBS and treatment with 10 units RNase ONE™ Ribonuclease (Promega; United States) for 30 min at room temperature. RNase reaction was stopped by adding 10 units RiboLockRNAse Inhibitor (Thermo Fisher Scientific; United States) to the mixture and incubation for 10 min at room temperature. Afterwards the isolation was performed according to a modified manufacture protocol. RNA concentration was measured by NanoDrop 1000 (Thermo Fisher Scientific; United States). MiRNA of urine exosomes was isolated according to the protocol of cells and in vitro exosomes.
TotalRNA of formalin-fixed paraffin-embedded (FFPE) primary bladder tumors was isolated using miRNeasy FFPE Kit (Qiagen, Germany). 20 sections of each tumor were prepared on a microscope slides to perform a macro-dissection of tumor areas. Tumor sections were transferred into a reaction tube. Afterwards, isolation of totalRNA was performed according to the manufacture protocol and RNA concentration was measured by NanoDrop 1000 (Thermo Fisher Scientific; United States).