Arabidopsis Transcriptome Profiling by RNA-seq

Total RNA was isolated from 12-day-old Arabidopsis seedlings using TRI Reagent (NRC), and DNA was removed by DNase I (Roche) treatment. PolyA RNA was then enriched from 1 μg of DNase I-treated RNA using Oligo d(T)25 Magnetic Beads (NEB S1419S), followed by RNA-seq libraries construction using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB E7420). RNA libraries were sequenced on an Illumina NovaSeq 6000 platform (PE150 bp), and the sequencing data were analyzed using the pRNASeqTools v.0.8 pipeline. Briefly, raw reads were aligned to the Arabidopsis genome (TAIR 10) using STAR v2.7.6a [56 (link)] with parameters ‘—alignIntronMax 5000—outSAMmultNmax 1—outFilterMultimapNmax 50—outFilterMismatchNoverLmax 0.1’, and counted by featureCounts v2.0.0 [57 (link)]. Normalization was performed by calculating the FPKM (Fragments Per Kilobase Million) for each gene, and differential gene expression analysis was conducted by DESeq2 v1.30.0 with a fold change of 1.5 and adjusted P value < 0.05 as the parameters [55 (link)].