RNA-Seq Experiment and Analysis

Approximately 30 mg of 7-d–old Arabidopsis seedlings were harvested and their total RNA was extracted using the MagMAX-96 Total RNA Isolation kit (AM1830; Ambion) according to manufacturer’s instructions. Library preparation was performed using 1 μg of high integrity total RNA (RNA integrity number > 8) using the TruSeq Stranded mRNA library preparation kit (cat. no. RS-122-2103; Illumina) according to the manufacturer’s instructions. The libraries were sequenced on a NextSeq 500 platform (Illumina).

For bioinformatics analysis, we first assessed the quality of reads using the program FastQC (www.bioinformatics.babraham.ac.uk/projects/fastqc/). Potential adaptor contamination and low quality trailing sequences were removed using the tool Trimmomatic (Bolger et al., 2014), before alignment to The Arabidopsis Information Resource 10 transcriptome using the software TopHat (Trapnell et al., 2009). Potential optical duplicates resulting from library preparation were removed using the Picard tools (https://github.com/broadinstitute/picard), and the read counts were normalized by the sample’s genome-wide reads coverage. Raw counts were determined by HTseq-count (Anders et al., 2015), and the software Cufflinks (http://cole-trapnell-lab.github.io/cufflinks/) was utilized to calculate Fragments Per Kilobase Million, which was then converted into TPM.