RNA-Seq of CHI treated Nb leaves
0.2g of leaves of the same size at the same position were cut with scissors and bagged into liquid nitrogen. Quality of total RNAs was verified on an Agilent 2100 Bioanalyzer and based on the rRNA ratio 25S/18S, RNA integrity number, and the absence of smear. The amplified fragments were sequenced using Illumina HiSeqTM 2500. Denovo assembly and differentially expressed gene analysis were described previously32. In brief, the raw reads obtained by the sequencing platform were performed quality control (QC) and filtered to obtain high-quality clean reads. After that, clean reads were aligned to the reference genome. After preforming the QC, follow-up analysis was performed, followed by quantitative analysis and structural analysis. Quantitative analysis includes quantification of genes, exons, and transcripts. Subsequent differential expression analysis was based on quantitative analysis, functional enrichment analysis, and time series analysis. Structural analysis consists of alternative splicing analysis, gene structure optimization, new transcript analysis, and SNP/InDel analysis, gene fusion analysis.