Viral dsRNA Extraction and Sequencing Protocol

CnFGV1 genomic dsRNA was extracted using the Double-RNA Viral dsRNA Extraction Mini Kit (iNtRON Biotechnologies, Seongnam-Si, Korea) and electrophoresed on a 1.5% (w/v) agarose gel. Potentially present fungal DNA and ribosomal RNA were eliminated treating the extracts with both enzymes dsDNase and S1 Nuclease (Thermo Fisher Scientific, Waltham, MA, USA). A subset of three dsRNA extracts (M10535, M10544, and M10545) was subjected to RNA-seq using the TruSeq RNA Sample Prep Kit (Illumina, San Diego, CA, USA) and sequenced on an Illumina MiSeq v2 (Microsynth AG, Balgach, Switzerland). De novo assembly of reads was carried out using Trinity v2.6.5 [39 (link)]. The obtained contigs were aligned using CLC Main Workbench v7 (CLC bio, Qiagen Digital Insights, Hilden, Germany) and subjected to searches using the ORFfinder resource of NCBI (https://www.ncbi.nlm.nih.gov; accessed on 22 July 2021) and the BLASTp suite of the UniProt portal (v 2.9.0+; https://www.uniprot.org; accessed on 22 July 2021).

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Cornejo C., Hisano S., Bragança H., Suzuki N, & Rigling D. (2021). A New Double-Stranded RNA Mycovirus in Cryphonectria naterciae Is Able to Cross the Species Barrier and Is Deleterious to a New Host. Journal of Fungi, 7(10), 861.

Publication 2021

dsDNase S1 Nuclease TruSeq RNA Sample Prep Kit Illumina MiSeq v2 CLC Main Workbench v7

Agarose Dsrna Enzymes Fungal dna Genomic Intron Qiagen Ribosomal rna Rna seq Rna viral

Corresponding Organization : Swiss Federal Institute for Forest, Snow and Landscape Research

Other organizations : Okayama University

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Variable analysis

independent variables

Use of the Double-RNA Viral dsRNA Extraction Mini Kit (iNtRON Biotechnologies, Seongnam-Si, Korea) to extract CnFGV1 genomic dsRNA
Treating the extracts with dsDNase and S1 Nuclease (Thermo Fisher Scientific, Waltham, MA, USA) to eliminate potentially present fungal DNA and ribosomal RNA

dependent variables

Presence and/or quantity of CnFGV1 genomic dsRNA in the extracts

control variables

Agarose gel concentration (1.5% w/v) used for electrophoresis
RNA-seq using the TruSeq RNA Sample Prep Kit (Illumina, San Diego, CA, USA) and sequencing on an Illumina MiSeq v2 (Microsynth AG, Balgach, Switzerland)
De novo assembly of reads using Trinity v2.6.5
Alignment of obtained contigs using CLC Main Workbench v7 (CLC bio, Qiagen Digital Insights, Hilden, Germany)
Searches using the ORFfinder resource of NCBI and the BLASTp suite of the UniProt portal (v 2.9.0+)

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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