Figure 4
From: Optimized cDNA libraries for virus-induced gene silencing (VIGS) using tobacco rattle virus
Schematic representation of VIGS-cDNA library synthesis method. Magnetic bead linked oligo(dT) primers are used to immobilize mRNAs and prime cDNA synthesis. RsaI digestion yielded short (~600 bp) fragments lacking 3' homopolymeric regions. Suppression subtractive hybridization (SSH) was used to subtract cDNA fragments present in a driver population of cDNAs from those present in a tester population [23, 24]. After ligation into a TRV-RNA2 vector, and amplification in E. coli, the cDNA inserts are transformed into A. tumefaciens to produce a collection of cDNAs suitable for both VIGS and EST sequencing.