Total RNA was extracted using Trizol reagent (#AG21102, Accurate Bioeng. Co., Ltd., Hunan, China) and the mRNA was purified using the Dynabeads® mRNA Purification Kit Dynabeads Oligo(dT)25 (#61002, Invitrogen). mRNA was treated by DNaseⅠ buffer (AM8170G, Thermo Fisher Scientific) and Turbo DNase (Thermo Fisher Scientific, AM2238) at 37 ℃ for 10 min and purified by ethanol precipitation to remove genomic DNA. Nanopore direct RNA sequencing libraries were prepared using about 1000ng mRNA following the SQK-RNA002 kit protocol (version: DRS_9080_v2_revK_14Aug2019). DRS library was added to the flow cell for sequencing on the Mk1C and GridION platform.