design: A synthetic DNA oligonucleotide template was in vitro-transcribed using HiScribe T7 High Yield RNA Synthesis Kit (NEB, E2040S) and purified with RNA Clean & Concentrator Kit-25 (Zymo Research, R1017). The IVT product was polyadenylated with E-PAP Poly(A) Tailing Kit (Thermo Fisher Scientific, AM1350) according to the manufacturer's protocol and cleaned using RNA Clean & Concentrator Kit-5 (Zymo Research, R1016). The library was prepared with SQK-RNA004 sequencing kit (Oxford Nanopore Technologies) from 500 ng of the RNA. Samples were loaded onto the primed PromethION RNA flow cell (ONT, FLO-PRO004RA). The direct RNA sequencing(DRS) was performed by PromethION 2 Solo device (ONT).  The DRS sequencing data was basecalled with Dorado (ver. 0.7.3) with rna004_130bps_sup@v5.0.0 basecalling model and options dorado basecaller --reference --chunksize 12000 --min-qscore 0 --emit-moves --estimate-poly-a. BAM files generated by Dorado were converted to FASTQ format using SAMTools (ver. 1.22.1).