RNA-Seq of VIGS tomato fruit
Total RNA was extracted from the fruit pericarp of TRV2-Empty control and TRV2-TDR4 silencing plants (three biology replicates each) using an RNeasy MiniKit (QIAGEN). RNA integrity was monitored on 1 % agarose gels stained with ethidium bromide (EB). RNA concentration was measured using Qubit RNA Assay Kit in Qubit 2.0 Flurometer (Life Technologies, CA, USA). Sequencing cDNA libraries were generated using NEBNext UltraTM RNA Library Prep Kit for Illumina (NEB, USA) following manufacturers instructions. In short, mRNA was enriched using oligo(dT)-attached magnetic beads. Fragmentation was done by divalent cations in NEBNext First Strand Synthesis Reaction Buffer. These fragments were used to synthesize first strand cDNA using random hexamer primer and M-MuLV Reverse Transcriptase. Then second strand cDNA synthesis was achieved using DNA Polymerase I and RNase H. Exonuclease/polymerase activities were used to convert overhangs into blunt ends. In order to select cDNA fragments with right size, the library fragments were purfied with AMPure XP system (Beckman Coulter, Beverly, USA). USER Enzyme (NEB, USA) was subsequently used with size-selected, adaptor-ligated cDNA. Then PCR was carried out with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. Eventually, PCR products were purified and library quality was measured on the Agilent Bioanalyzer 2100 system. According to the manufacturers instructions, the clustering of the index-coded samples was performed on a cBot Cluster Generation System using HiSeq 4000 PE Cluster Kit (Illumia). Then the library preparations were sequenced on an Illumina Hiseq 4000 platform and 150bp paired-end reads were generated.