Dnase 1

RNA isolation and sequencing of poplar roots and leaves were performed as described previously74 (link). Briefly, total RNA was isolated from the pooled root or leaf powder as described above using a plant RNA extraction kit (R6827, Omega Bio-Tek, GA, USA), i.e., six total RNA samples were obtained for each tissue within each treatment. Genomic DNA in the RNA extract was digested using DNase I (E1091, Omega Bio-Tek, GA, USA). Next, equal amounts of total RNA from each preparation of each tissue within each treatment were pooled for subsequent library construction and RNA sequencing, i.e., one library was established per tissue for each treatment and used for RNA sequencing. Library construction and Illumina sequencing were performed by Shanghai Biotechnology Corporation (Shanghai, China). The libraries obtained for the roots and leaves were sequenced by using Illumina Genome Analyzer HiSeq 2500 and HiSeq 2000 systems, respectively, thereby obtaining single-end reads measuring 50 bp length, which exhibited the visible difference in raw reads between the roots and leaves (Table S1). High quality reads that passed the Illumina quality filters were selected for further sequence analysis. The sequencing datasets are available at NCBI Sequence Read Archive (SRA, http://www.ncbi.nlm.nih.gov/Traces/sra/, accession number: SRP064930).

Double-stranded RNAs were extracted as described previously [32 (link)], with the following modifications: The extracted dsRNAs were not incubated with oligo d(T)25 magnetic beads and 2× LTE buffer was replaced with 2× STE (500 mM NaCl; 20 mM Tris-HCl, pH 8.0; 30 mM EDTA, pH 8.0). Single-stranded RNAs were extracted from yeasts using the RNeasy RNA extraction kit (Qiagen) with the bead beating method and an on column DNase I digestion. After column elution, an additional incubation with DNase I (Omega Bio-Tek) was performed at 37°C for 10 min followed by 75°C for 10 min when the removal of additional contaminating DNAs was required.