DRS of apple
Nanopore direct RNA sequencing was performed by Benagen Technology Co., Ltd (Wuhan, China). The total RNA from WT and A10 leaves was extracted using the Plant RNA Kit (Omega, R6827), and RNA concentration and quality were measured using a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE) and Qubit 3.0 Fluorometer (Life Technologies, Carlsbad, CA, USA). Each total RNA sample (50 g) was processed with mRNA Dynabeads enrichment kit (Thermo, 61006) to enrich mRNA. Then the Nanopore RT Adapter was ligated to mRNA using T4 DNA ligase (NEB-M0202M). The mRNA added with RT adapter was reverse-transcribed, and the reverse transcription product was purified with Agencourt RNAClean XP beads, then RNA adapter was added, and the ligation product was purified with Agencourt RNAClean XP beads (Beckman, California, USA) again. Finally, the prepared library was loaded into the R9.4 sequencing chip, and sequenced with the PromethION sequencer (Oxford Nanopore Technologies, Oxford, UK) for 48-72h. Three independent biological replicates were sequenced to generate real-time single-molecule sequencing. The raw data of Nanopore sequencing was converted from the fast5 format to the fastq format after base calling with GUPPY 3.2.6 software.