dsRNA sequencing of Verticillium albo-atrum partitivirus 1: Verticillium nonalfalfae strain 256VD
The first step in the search for viral-infected fungi is to look for the presence of dsRNAs, as all RNA viruses have a dsRNA stage during their infection cycle. To isolate dsRNA, fungi were cultured on cellophane membranes overlaying a half Czapek Dox plate (supplemented with 1g/l of malt extract, 1 g/l of yeast extract, 1g/l of peptone, and 15 g/l of agar) at room temperature. Harvested mycelia were homogenized with a pestle and mortar in the presence of liquid nitrogen. The dsRNA was extracted using cellulose C6288 and analyzed by electrophoresis on 1% agarose gel. The nature of the dsRNA was confirmed by digestion of the extracts with DNase I (New England BioLabs) and S1 nuclease (Thermo Fisher Scientific). Libraries of dsRNAs were constructed using the Ion Total RNA-Seq Kit v2 (Thermo Fisher Scientific) and were barcoded using the Xpress RNA-Seq Barcode 116 Kit (Thermo Fisher Scientific) according to the manufacturers instructions. The yield and size distribution of the amplified cDNA libraries were determined using the Agilent 2100 Bioanalyzer (Agilent Technologies). Sequencing was performed on the Ion GeneStudio S5 Prime System (Thermo Fisher Scientific) and Ion PGM Sequencer (Thermo Fisher Scientific).