Three biological replicate samples were collected from the aerial portions of WT and BR1OE mutant plants 25 days post-planting. After snap freezing using liquid nitrogen, these samples were stored at −80°C. RNA extraction was performed as detailed previously (Jiang et al., 2018 (link)), and 1% agarose gel electrophoresis was used to detect any RNA contamination or degradation while a NanoPhotometer® instrument (IMPLEN, CA, USA) was used to confirm RNA purity. A Qubit® RNA Assay Kit and a Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) were used to quantify the RNA concentrations in individual samples, while an RNA Nano 6000 Assay Kit and a Bioanalyzer 2100 instrument (Agilent Technologies, CA, USA) were used to confirm RNA integrity. Sequencing libraries were prepared from 1 μg of RNA per sample with a NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) based on provided directions. Library sequencing was then performed with an Illumina Hiseq platform to generate 125 bp/150 bp paired-end reads.
Initial data were filtered with Fastp (v0.19.3) to remove adapter-containing reads, reads containing > 10% N bases, and reads with > 50% low-quality (Q ≤ 20) bases. The clean reads were then compared to the Arabidopsis TAIR10 genome which was downloaded from The Arabidopsis Information Resource (TAIR) (https://www.arabidopsis.org/) using HISAT (v2.1.0). New gene predictions were made using StringTie (v1.3.4d), while gene alignment was calculated with FeatureCounts (v1.6.2), and fragments per kilobases of exons per million mapped reads (FPKM) expression values were then calculated for all transcripts. Differentially expressed genes (DEGs) were identified using DESeq2 (v1.22.1) based on Benjamini & Hochberg-corrected p-values, a |log2Fold Change| ≥ 1, and a false discovery rate (FDR) < 0.05. Hypergeometric tests were used for Gene Ontology (GO) term and KEGG pathway enrichment analyses. Gene expression was validated using primer pairs listed in Supplementary Table S1.
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