Nanopore Direct RNA sequencing of in vitro transcribed 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. 100 ng of HEK293T poly(A)+ RNA was combined with the VN primer and the Strand-switching primer. The primers had sequences identical to the cDNA-PCR Sequencing Kit (ONT, SQK-PCS109) and were manufactured by Bionics. For reverse transcription, the poly(A)+ RNA and primer mix was incubated with Maxima H Minus Reverse Transcriptase (Thermo Scientific, EP0751) at 42 C for 90 min, 85 C for 5 min, and held at 4 C. The first-stranded cDNA was amplified using LongAmp Taq 2X Master Mix (NEB, M0287S) and IVT_T7_Forward and Reverse primers as follows: initial denaturation step at 95C for 30 s (1 cycle); denaturation at 95C for 15 s, annealing at 62C for 15 s, and extension at 65C for 15 min (10 cycles); final extension at 65C for 15 min (1 cycle); and a hold at 4C. To eliminate any remaining single-stranded DNA, the amplified cDNA was treated with Exonuclease I (NEB, M0293S) and purified with AMPure XP beads (Beckman Coulter) according to the manufacturer's guidelines. The purified HEK293T cDNA 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. 1.16.1).
SUB15084387
ON0105