Howard Hughes Medical Institute, University of California Santa Cruz, Santa Cruz, California, USA.
Nat Methods. 2010 Dec;7(12):995-1001. doi: 10.1038/nmeth.1529. Epub 2010 Nov 7.
Classical approaches to determine structures of noncoding 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 developed fragmentation sequencing (FragSeq), a high-throughput RNA structure probing method that uses high-throughput RNA sequencing of fragments generated by digestion with nuclease P1, which specifically cleaves single-stranded nucleic acids. In experiments probing the entire mouse nuclear transcriptome, we accurately and simultaneously mapped single-stranded RNA regions in multiple ncRNAs with known structure. We probed in two cell types to verify reproducibility. We also identified and experimentally validated structured regions in ncRNAs with, to our knowledge, no previously reported probing data.
传统的方法是使用酶和化学物质一次探测一种 RNA，通过凝胶电泳来识别反应位置，从而确定非编码 RNA（ncRNA）的结构。为了加速 RNA 结构推断，我们开发了片段测序（FragSeq），这是一种高通量 RNA 结构探测方法，它使用核酸酶 P1 消化产生的片段的高通量 RNA 测序，该酶专门切割单链核酸。在探测整个小鼠核转录组的实验中，我们准确地同时映射了多个具有已知结构的 ncRNA 中的单链 RNA 区域。我们在两种细胞类型中进行了探测以验证重现性。我们还在 ncRNA 中鉴定和实验验证了具有结构的区域，据我们所知，这些区域以前没有报道过探测数据。
