title: Graminella nigrifrons Transcriptome or Gene expression
design: G. nigrifrons fed on MCDV- and MFSV. Each replication consisted of three treatments, feeding on MFSV-infected, MCDV-infected, or healthy maize, with two time points, 4 h or 7 d per treatment. 240 newly emerged virgin females (≤ 24 h after molt from 5th instar nymph stage) were randomly chosen in roughly equal numbers from three maintenance cages, and starved for 3 h. For each treatment/time combination, 40 females were divided evenly into two 0.1 m3 cage each containing one 3.5 – 4 wk old MFSV-infected, MCDV-infected, or healthy (control) maize plant. After 4 h or 7 d, the females from each cage were collected, frozen in liquid nitrogen, and stored at -80C until RNA isolation. Each treatment/time point was replicated four times using nymphs from different cohorts (24 samples total).  RNA isolation and cDNA library preparation. Total RNA was extracted from the pools of 25 leafhoppers using the RNeasy Mini Kit (QIAGEN, Germantown, MD). RNA quantity and quality was assessed using the Experion Automated Electrophoresis System (Bio-Rad, Hercules, CA). Only samples with RNA Quality Indicator values ≥10 were selected for cDNA synthesis.  RNA (800 ng per sample) was used to generate adaptor-ligated double-stranded cDNA libraries for RNA-Seq using the TruSeq Sample Prep Kit V1 and V2 (Illumina, San Diego, CA) following the manufacturer’s protocol. Quantification and quality inspection of ds-cDNA was carried out using the Experion Automated Electrophoresis System (Bio-Rad, Hercules, CA) and Qubit® 2.0 Fluorometer (Life Technologies, Carlsbad, CA). Samples were diluted to 20 nM and pooled to generate the multiplexed cDNA library (24 adaptor-tagged sample pools total: 3 treatments x 2 time points x 4 replicates).  Transcriptome sequencing and assembly. The cDNA library (50 fmoles) was sequenced on one flow cell lane using the Illumina HiSeqTM2000 platform at the Ohio State University Comprehensive Cancer Center. Four fluorescently labeled nucleotides and a specialized polymerase were used to determine the clusters base by base in parallel. The mean library insert sequence size was 272 bp and both ends of the library were sequenced to generate 100 bp raw paired end reads. Illumina Analysis Package CASAVA 1.8.2 was used to perform bcl conversion and demultiplexing. Image deconvolution and quality value calculations were carried out using the Illumina GA pipeline v1.6.