Nanopore Direct RNA sequencing of synthetic RNA oligonucleotide containing m6A, replicate 1
Building blocks (BBs), that are chemically synthesized RNA oligonucleotides, were purchased from IDT. The quality of the BBs was checked by PAGE: 1/10x aliquots of BBs were analyzed on 12% TBE-Urea gel system (National Diagnostics, SequaGel UreaGel System) with 20/100 single-stranded oligo length standards (IDT). We generated longer RNA molecules for sequencing by ligating the BBs iteratively. 640 pmol of 87-nt BBs were liagted by incubating with 19% PEG8000, 1 mM ATP, 10% DMSO, 1x T4 RNA ligase buffer (NEB, B0216S), and 50 U T4 RNA ligase 1 at 33 C for 16 hrs. For every 100l solution, ligated RNA was isolated using guanidium thioisocyanate-phenol-chloroform (Trizol) extraction using 300 l of QIAzol Lysis Reagent (Qiagen) and 120 l chloroform (Sigma-Aldrich), and purified with RNA Clean & Concentrator-5 (Zymo Research, R1013) according to the manufacturer's protocol. We selected RNA molecules longer than 300 nts for sequencing. The purified BB ligates were separated using an 8% TBE-Urea gel system (National Diagnostics) (Extended Data Fig. 2a). Gel slices containing RNAs longer than 300 nts were obtained and eluted with 3x volume of 300 mM NaCl buffer overnight at 4 C. To remove the gel debris, the eluted samples were purified with 0.22 m Spin-X Centrifuge Tube Filter (Corning). RNAs were recovered by Oligo Clean & Concentrator (Zymo Research, D4061) following the manufacturer's instructions. The ligated BBs were polyadenylated to allow sequencing via DRS. E-PAP Poly(A) Tailing Kit (Thermo Fisher Scientific, AM1350) was used for polyadenlyation. 30 pmol of ligated RNA was incubated with 1x E-PAP buffer, 2.5 mM MnCl2, 10 M ATP, 100 U RNaseOUT recombinant ribonuclease inhibitor (Invitrogen, 10777019), and 8 U E-PAP in 50 l solution at 37 C for 45 min. The product was purified with Oligo Clean & Concentrator (Zymo Research, D4061). To remove circular RNAs and enrich poly(A)-tailed linear RNA, the purified RNAs were treated with Dynabeads Oligo(dT)25 (Invitrogen, 61002) following the manufacturer's protocol. 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 --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).
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