cDNA_randomPriming of mycovirus in Fusarium Oxysporum f. sp. Cubense Tropical Race 4
RNA sample purity was first determined using 1% agarose gel electrophoresis to look for possible contamination and degradation. RNA purity and concentration were then examined using NanoPhotometer spectrophotometer. RNA integrity and quantity were finally measure using RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system. RNA library for meta-transcriptomics sequencing was prepared as rRNA depletion and non-stranded method. The ribosomal RNA was depleted from total RNA using the appropriate rRNA removal kits following manufacturer's instruction. NEBNext Ultra II RNA Library Prep Kit for Illumina was used to prepare libraries. RNA was first fragmented into 250~300 bp fragments and subsequently reverse-transcribed into cDNA using random hexamers. Remaining overhangs of double-strand cDNA was converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, sequencing adaptors were ligated to the cDNA. In order to select cDNA fragments of preferentially 250~300 bp in length, the library fragments were purified with AMPure XP system (Beverly, USA). Amplification of cDNA was performed using PCR. After library construction, the concentration of library was measured by the Qubit fluorometer and adjusted to 1ng/L. Agilent 2100 Bioanalyzer (Agilent, USA) was deployed to examine the insert size of the acquired library. At last, the accurate concentration of cDNA library was again examined using qPCR. Once the insert size and concentration of the library has been determined, the sample was sequenced on Illumina NovaSeq6000 with a sequencing configuration of PE150.