Direct nanopore sequencing of modified IVT RNA
The MEGAscript T7 Transcription Kit (ThermoFisher, cat # AM1334) was used for vitro transcription (IVT) in the presence of varying amounts of modified nucleotides. In vitro transcribed RNAs were purified from their reaction mixes using RNAClean XP beads (Beckman-Coulter, cat# A63987), and the integrity of the RNA was verified using the Agilent RNA6000 Nano Kit (cat# 5067-1511). IVT RNAs were 3-polyadenylated using Oxford Nanopores protocol for polyadenylating with E. coli poly(A) polymerase, followed by Oxford Nanopores protocol for direct RNA sequencing (SQK-RNA002). RNA samples were sequenced using an Oxford Nanopore MinION Mk1B device or a five-cell GridION sequencer, with FLO-MIN106D flow cells (which use the v9.4.1 pore).