Scheme of in-depth transcriptome profiling for alternative splicing in response to flg22. Two-week-old plate-grown wild-type (WT) Col-0 and cpl3-3 mutant seedlings treated with H 2 O (mock) or 100 nM flg22 for 60 min were subjected to RNA isolation and sequencing. To capture the transcripts with low abundance, RNA-seq was performed with Illumina HiSeq 2500 to obtain 120 million reads per sample with the paired-end 150-bp read length, which is about 530 coverage of Arabidopsis transcriptome. The sequenced reads were aligned to the AtRTD3 reference transcriptome for quantification. The 3D RNA-seq analysis was performed to identify differentially expressed genes (DEGs), differentially alternatively spliced genes (DASs), and transcripts with differential transcript usage (DTUs). The bottom left panel shows an example of DEGs with two differentially expressed transcripts, where changes in abundance between conditions (dH 2 O and flg22 treatments) are measured by log 2 fold change. Total gene expression is represented in the blue line, which is the sum of the expression of all individual transcripts (green and yellow lines). The percentage values denote the expression ratios of individual transcripts relative to the total gene expression. The bottom right panel illustrates examples of DAS and DTU. For a DAS gene, it must have more than one transcript, and changes in expression between individual transcripts (green, yellow, and purple lines) and the total gene expression (blue line) are compared between conditions. The change in percent spliced (ΔPS) is the percentage change in the abundance of a transcript relative to the total gene expression. For a gene to be classified as DAS, at least one transcript has a |ΔPS|≥ 0.1. In DTU analysis, individual transcripts show different expression patterns compared to other transcripts of the same gene. DTU is identified by comparing the change in expression of each transcript to the average expression change of the other transcripts within the same gene. In this example, the transcripts represented with green and yellow lines, but not with the purple line, are DTUs.
