RNA-Sequencing of virus species infecting sweetpotato
Total RNA of each sample was extracted using TRIzol Reagent (Invitrogen)/RNeasy Mini Kit(Qiagen)/other kits. Total RNA of each sample was quantified and qualified by Agilent 2100 Bioanalyzer (AgilentTechnologies, Palo Alto, CA, USA), NanoDrop (Thermo Fisher Scientific Inc.) and 1% agarose gel. 1 g totalRNA with RIN value above 7 was used for following library preparation.Next generation sequencing library preparations were constructed according to the manufacturersprotocol (NEBNext Ultra RNA Library Prep Kit for Illumina). The poly(A) mRNA isolation wasperformed using NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB) or Ribo-Zero rRNA removalKit (illumina). The mRNA fragmentation and priming was performed using NEBNext First Strand SynthesisReaction Buffer and NEBNext Random Primers. First strand cDNA was synthesized using ProtoScript IIReverse Transcriptase and the second-strand cDNA was synthesized using Second Strand SynthesisEnzyme Mix. The purified double-stranded cDNA (by AxyPrep Mag PCR Clean-up (Axygen)) was thentreated with End Prep Enzyme Mix to repair both ends and add a dA-tailing in one reaction, followed bya T-A ligation to add adaptors to both ends. Size selection of Adaptor-ligated DNA was then performedusing AxyPrep Mag PCR Clean-up (Axygen), and fragments of ~360 bp (with the approximate insert sizeof 300 bp) were recovered. Each sample was then amplified by PCR for 11 cycles using P5 and P7 primers,with both primers carrying sequences which can anneal with flow cell to perform bridge PCR and P7primer carrying a six-base index allowing for multiplexing. The PCR products were cleaned up usingAxyPrep Mag PCR Clean-up (Axygen), validated using an Agilent 2100 Bioanalyzer (Agilent Technologies,Palo Alto, CA, USA), and quantified by Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA).Then libraries with different indices were multiplexed and loaded on an Illumina HiSeq instrumentaccording to manufacturers instructions (Illumina, San Diego, CA, USA). Sequencing was carried outusing a 2x150bp paired-end (PE) configuration; image analysis and base calling were conducted by theHiSeq Control Software (HCS) + OLB + GAPipeline-1.6 (illumina) on the HiSeq instrument.