Laodelphax striatellus that is not infected with rice stripe virus (Naive)
The double strand cDNA was synthesized from two RNA pools according to Ng’s full-length cDNA synthesis protocol [40] with modification. A GsuI-oligo dT primer was used to prime first-strand cDNA synthesis from 20µg of mRNA, using 1000 units of Superscript II reverse transcriptase (Invitrogen). Then diol group of the CAP structure of mRNA was oxidized by NaIO4, followed by biotinylation with the biotin hydrazide (long arm, Vector Laboratories). After RNaseONE digestion, Dynal M280 streptavidin Dynabeads were used to select biotinylated RNA/cDNA. The first-strand cDNA was then released by alkaline hydrolysis, and two 5’ adaptors (N5 and N6 adaptors) were ligated to the 5’-end of the first-strand cDNA. Double strand cDNA was synthesized by primer extension using Ex Taq polymerase (TaKaRa). PolyA tail was removed by GsuI digestion, and cDNA size fractionation was performed using cDNA size fractionation column (Invitrogen). Each cDNA fraction larger than 800bp was sonicated to the range of 300-800 bp, then pooled together with the other cDNA samples ranging from 300 bp to 800 bp. The prepared cDNA were transformed into single-stranded template DNA (sstDNA) libraries by using the GS DNA Library Preparation kit (Roche Applied Science), sstDNA libraries were clonally amplified in a bead-immobilized form by using the GS emPCR kit (Roche Applied Science) and sequenced on the 454 Genome Sequencer FLX instrument.