Long-read transcriptome-wide RNA structure maps using DMS-FIRST-seq
Begik, O.; Diensthuber, G.; Borovska, I.; Mattick, J. S.; Incarnato, D.; Novoa, E. M.
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Coupling chemical probing with nanopore direct RNA sequencing (dRNA-seq) has been proposed as a promising approach to capture RNA structural dynamics at single-molecule resolution. However, dRNA-seq 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 overcomes these limitations by combining chemical probing with nanopore cDNA sequencing to achieve single nucleotide resolution. By systematically assessing various RT enzymes and buffers, we optimize conditions that minimize drop-off and enhance error signatures linked to RNA structure. Our results demonstrate that DMS-FIRST-seq generates high-resolution, accurate RNA structure maps in both in vivo and in vitro contexts, thus providing a simple and cost-effective method for transcriptome-wide isoform-specific RNA structural analyses.
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