*4.6. RNA-seq and Analysis*

Twenty-day-old transgenic and WT *A. thaliana* seedlings were treated with 50 μM CdCl2 for 24 h and sampled for RNA extraction [50]. RNA-seq libraries were created following the manufacturer's instructions for the NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, Ipswich, MA, USA). The cDNA fragments, ranging in length from 250 to 300 bp, were screened using AMPure XP system (Beckman Coulter, Beverly, CA, USA). Illumina Hiseq 2000 (Illumina, San Diego, CA, USA) was utilized for library sequencing and paired-end read production.

Genes were assembled using the full-length transcript sequence as a reference [51]. The FPKM value obtained from read counts was converted to a TPM value to obtain the expression level of each isoform. Based on analysis using differential expression analysis software DESeq2, genes with padj < 0.05 and |log2foldchange| > 1 were identified as differentially expressed genes (DEGs). GO enrichment analysis was performed on DEGs using Wallenius' noncentral hypergeometric distribution of the GOseq R package. The KEGG enrichment analysis was performed using KOBAS software.
