Small-RNA sequencing of WUS-induced GFP RNA degradation in Nicotiana benthamiana leaves

The 35S::GFP fluorescence was silenced 6-day after infiltration to Nicotiana benthamiana leaves due to the post-transcriptional gene silencing, but became stable by adding the viral suppressor 2b which repressed RNA silencing in plants. We performed the small RNA high throughput sequencing to test whether WUS can inhibit 2b functions in repressing plant RNA silencing. Overall design: The abaxial surface of leaves in 3-week-old N. benthamiana were infiltrated with the mixture of Agrobacterium tumefaciens harbored different constructs, including 35S::GFP + 35S::GUS, 35S::GFP + 35S::2b + 35S::GUS, 35S::GFP + 35S::2b + 35S::WUS. Six days after infiltration, the leaves were used to isolate the small RNA with two biological replicates. Small RNA high-throughput sequencing data was cleaned by removing adaptor and low quality reads. The 18-30 nt reads were mapped with GFP sequence to analysis the total reads with different length in each samples.