FragSeq: transcriptome-wide RNA structure probing using high-throughput sequencing

Previous efforts to determine structures of non-coding RNA (ncRNA) probed only one RNA at a time with enzymes and chemicals, using gel electrophoresis to identify reactive positions. To accelerate RNA structure inference, we have developed FragSeq, a high-throughput RNA structure probing method that uses high-throughput RNA sequencing on fragments generated by nuclease P1, which specifically cleaves single stranded nucleic acids. In experiments probing the entire mouse nuclear transcriptome, we show that we can accurately and simultaneously map single-stranded regions (ssRNA) in multiple ncRNAs with known structure. We carried out probing in two cell types to demonstrate reproducibility. We also identified and experimentally validated structured regions in ncRNAs never previously probed.