Wheat Transcriptome Sequencing via Nanopore

For library construction (Figure 5C), DNA contamination was removed from total RNA extracted using the RapidOut DNA Removal Kit (Thermo Fisher Scientific, Waltham, MA, USA). QIAseq FastSelect rRNA Plant Kit (Qiagen, Hilden, Germany) was used to remove wheat rRNA (ribodepletion) and purified with AMPure XP beads (Beckman Coulter, Brea, CA, USA). The purified RNA concentration was measured using a Qubit 4 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). Libraries were prepared using the Direct cDNA Sequencing Kit (SQK-DCS109; Oxford Nanopore Technologies, Oxford, UK) following the manufacturer’s instructions and previous studies [31 (link),53 (link)]. The prepared libraries were loaded on one Flongle Flow Cell (FLO-FLG001, R9.4.1; Oxford Nanopore Technologies, Oxford, UK) per sample, and the sequencing run was started on a MinION device with a Flongle adapter (Oxford Nanopore Technologies, Oxford, UK). Sequencing was performed for 24 h using MinKNOW software (version 21.11.7; Oxford Nanopore Technologies, Oxford, UK) and base-calling in High-accuracy mode with Guppy (version 5.1.12; Oxford Nanopore Technologies, Oxford, UK). Sequencing adapters were removed with Geneious Prime (version 21.1.1). Viruses were identified in ‘What’s in my Pot?’ (WIMP) workflow of EPI2ME Agent (version 3.4.2) and NCBI BLASTn using FASTQ files generated by the ONT platform.