RNA Extraction and Sequencing from Plant Material

RNA preparation and sequencing was performed as described previously [42 (link)]. Briefly, approximately 100 mg of plant material (5 leaf discs) was used for RNA extraction using the Qiagen RNeasy Plant Mini kit (Qiagen, Hilden, Germany), according to the manufacturer’s protocol, with an added DNase treatment interruption step. The DNase treatment was performed on column using the Invitrogen PureLink™ DNase set (Thermo Fisher Scientific, Massachusetts, USA) according to the manufacturer’s protocol. The RNA quality and concentration were both corroborated by ND-1000 NanoDrop and by a 2100 Bioanalyzer using RNA Nano chips (Agilent Technologies, CA, USA). Polyadenylated messenger RNA was captured from 200 ng total RNA per sample using magnetic beads and Illumina adaptors with sample-specific barcode sequences that were ligated before subsequent library amplification using PCR via the Illumina TruSeq RNA poly-A selection kit. Sequencing of 150 bp paired-end libraries was carried out using the Illumina NovaSeq6000 S4 platform (SciLifeLab, Stockholm, Sweden). All raw sequencing data in this study have been deposited in National Center for Biotechnology Information (NCBI) under BioProject accession number PRJNA755645.