Nanopore Direct RNA sequencing of HEK293T poly(A)+ transcriptome
Total RNA was extracted with QIAzol, precipitated with isopropanol, and washed with ethanol. After DNase I treatment, RNA was purified with RNA Clean & Concentration kit-25 (Zymo Research, R1017). The quantity and quality of total RNA were measured by 4200 Tapestation system (Agilent). From 20 g of total RNA, poly(A) RNA selection was performed with Dynabeads Oligo(dT)25 according to the manufacturer's instruction. The library was prepared with SQK-RNA004 sequencing kit (Oxford Nanopore Technologies). 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. 1.16.1).
SUB15084387
ON0090