title: RNA-Seq(quantative) of thrips infected with TSWV
design: To prepare non-infected and infected F. occidentalis samples, thrips adults (about 3d after eclosion) were transferred to healthy or TSWV-infected D. stramonium plants, kept in a separate growth chamber for 24h(27 ± 1 °C, 16L: 8D ), in order to thrips ovipositing into plant. Then, healthy or TSWV-infected D. stramonium plants were placed in climate-chamber separately, removing thrips adult. After about 3d, thrips eggs in D. stramonium plant were hatched. The 1st instars of thrips were kept on healthy or TSWV-infected D. stramonium plants 48h, for fully acquiring TSWV. Then the 1rst instars were transferred to fresh bean pods without TSWV, for rearing to 2nd instar, pre-pupae, pupae, and adult. Then approximately 1,000 non-viruliferous and viruliferous 1rst instar, 2nd instar, pre-pupae and pupae, adults of F. occidentalis were collected, respectively. Therefore, there were eight F. occidentalis samples. Two biological replicates for all of the F. occidentalis samples were conducted and processed independently. One replicate was used in the gene expression profile analysis and Illumina sequencing, and the other was used for the quantitative PCR (qPCR) analysis. Total RNA was isolated using SV total RNA isolation system (Promega) according to the manufacturer’s protocol. To obtain complete gene expression information, a pooled RNA sample including different treatments and different developmental stages [TSWV infected and uninfected, 1rst instar, 2nd instar, pre-pupae and pupae, adult], except for egg [under the surface of plant tissues, not convenient to collect and can’t be infected by TSWV], was used for transcriptome analysis. RNA integrity was confirmed using the 2100 Bioanalyzer (Agilent Technologies) with a minimum RNA integrated number value of 8.Enrichment of mRNA, fragment interruption, addition of adapters, size selection and PCR amplification and RNA-Seq were performed by staff at Beijing Genome Institute (BGI) (Shenzhen, China). Poly (A) mRNA was isolated using oligo dT beads, and then broken into short fragments.  The single-end libraries of thrips TSWV infected(including pool of 1rst instar, 2nd instar, pre-pupae and pupae, adult) for comparison expression profiling between infected and non-infected thrips were prepared following Illumina’s protocols.