title: Comparative transcriptome analysis reveals crucial roles of pathogenesis-related genes and ethylene-responsive factor genes in response to Ralstonia solanacearum
design: R. solanacearum isolate CQPS-1 (race 1 biovar 3 phylotype I sequevar 17) was cultivated at 30°C. Tobacco (Nicotiana tabacum) cultivars PVH2254 and Yunyan87 were grown in a growth room at 28°C at 70% relative humidity with a 14-h photoperiod. R. solanacearum strain was grown in B liquid medium, for 16 h at 30°C with shaking. Bacteria were collected by centrifugation and resuspended in distilled water. Bacterial growth was assessed on tobacco by wounding root-inoculation with a 108 colony-forming units (CFU) ml-1 CQPS-1 inoculum and distilled water (as a negative control), respectively. Tissues of two tobacco cultivars were harvested at 30 mpi, 1, 3 and 5 dpi, respectively. Each sample included roots and base stems of 4 tobacco plants. Total RNA of each sample was extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). RNA concentration and integrity were analyzed on the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA). Tobacco cDNA libraries were generated using Illumina TruSeq™ RNA Sample Preparation Kit (Illumia, San Diego, USA) according to manufacturer’s recommendations. mRNA was purified from total RNA by poly-T oligo-attached beads. First strand cDNA was synthesized using random oligonucleotides and SuperScript II and second strand cDNA was performed using DNA Polymerase I and RNase H. Before sequencing, all 16 individual libraries were normalized and pooled together in a single lane on the Illumina HiSeq2000 platform. Paired-end reads were generated.