Long-read transcriptome-wide RNA structure maps using DMS-FIRST-seq

Transcriptome-wide RNA structure mapping has largely relied on coupling chemical probing reagents with high-throughput sequencing. More recently, the integration of chemical probing with nanopore direct RNA sequencing (DRS) has been proposed as a promising approach to capture RNA structural dynamics at single-molecule resolution.

However, DRS produces widespread rather than single-nucleotide signals from modified bases, thereby limiting its precision in RNA structure prediction. Here we introduce FIRST-seq, a method that couples chemical probing with nanopore cDNA sequencing to overcome these limitations, achieving single nucleotide resolution. By systematically examining various RT enzymes and buffers, we optimize conditions that minimize drop-off and enhance error signatures linked to RNA structure.