Differential Gene Expression Analysis by RNA-Seq

The leaf samples from three biological replicates from 4-week-old NbPDS- (different fragments shown in Fig. 6) or NbQM/RPL10-silenced N. benthamiana were harvested and immediately frozen in liquid nitrogen. Total RNA was extracted and sent to the Noble Research Institute Genomics Core Facility for analysis. RNA samples with high quality (RNA integrity >7.5, as assessed by the Agilent 2100 Bioanalyzer) were used for library preparation following the manufacturer's instructions (Illumina). Paired-end reads were generated from 21 libraries sequenced with Illumina 70 HiSeq 2000. After adapter sequences and low-quality reads were removed, the remaining reads were aligned to the annotated N. benthamiana reference transcriptome (Bombarely et al., 2012; https://solgenomics.net/organism/1490/view) and the gene-wise raw counts were calculated. The differential analysis was carried out using the DESeq tool (Anders and Huber, 2010). Genes with >2-fold change in expression and P < 0.05 were considered differentially expressed. Data from all of the 21 samples (seven treatments × three biological replicates) have been uploaded into our GEAUniversal gene expression atlas platform (https://bioinfo.noble.org/vigs/) for data normalization, visualization, and differential expression analysis using RNA-Seq analysis software (Li and Dewey, 2011; Love et al., 2014). The normalized expression data for all seven treatments can be directly visualized at https://bioinfo.noble.org/vigs/4100/transcript/profile/5?sessionid=vigs.