RNAseq of N. benthamiana: low geraniol (overexpression of GFP, CrGES), 5 dpi, replicate1
Leaf tissue from three biological replicates of each condition and a mock infiltrated control were collected five days post infiltration and flash-frozen in liquid nitrogen. Total RNA was isolated using the RNeasy plant mini kit (Qiagen) with recombinant DNase I (Roche) treatment. Libraries were constructed on a Sciclone G3 NGSx workstation (PerkinElmer, Waltham, MA) using the TruSeq RNA protocol v2 (Illumina 15026495 Rev.F). RNA quality was assessed using the Quant-iT RNA Assay Kit (Life technologies/Invitrogen Q-33140). 1 g of RNA was purified to extract polyadenylated mRNA using biotin beads, fragmented, and primed with random hexamers to produce first strand cDNA followed by second-strand synthesis to produce ds cDNA. Sample ends were blunted and A-tailed and indexing adapters with corresponding T-overhangs were ligated. The ligated products were size-selected and unligated adapters were removed using XP beads (Beckman Coulter A63880). Samples were amplified with a cocktail of PCR primers to the adapters. The insert size of the libraries was verified using the LabChipGX (PerkinElmer 5067-4626) and DNA High Sensitivity DNA reagents (PerkinElmer CLS760672). Equimolar quantities of each library were pooled, five libraries per pool, and sequenced on one lane of a HiSeq 2500 generating 100 base pair paired-end reads