design: Total RNA was extracted with QIAzol, precipitated with isopropanol, and washed with ethanol. After DNase I treatment, RNA was in vitro polyadenylated using E-PAP Poly(A) Tailing Kit (Thermo Fisher Scientific, AM1350). The polyadenylation was optimized from the manufacturer's protocol with minor changes. 2 g of total RNA was incubated with 1x E-PAP buffer, 2.5 mM MnCl2, 0.1 mM ATP, 10 U RNaseOUT recombinant ribonuclease inhibitor (Invitrogen, 10777019), and 1 U E-PAP in 10 l solution at 37 C for 10 min, and purified with Oligo Clean & Concentrator (Zymo Research, D4061). The DRS sequencing library was prepared from 500 ng of poly(A)-tailed total RNA using the SQK-RNA004 sequencing kit (ONT), following the manufacturer's protocol. 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 were 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).