期刊
NATURE PROTOCOLS
卷 10, 期 11, 页码 1643-1669出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2015.103
关键词
-
资金
- US National Institutes of Health (NIH) [AI068462]
- National Science Foundation [MCB-1121024, DGE-1144081]
- NIH training grant in molecular and cellular biophysics [T32 GM08570]
- Ruth L. Kirschstein NRSA Fellow [F32 GM010169]
- Direct For Biological Sciences
- Div Of Molecular and Cellular Bioscience [1121024] Funding Source: National Science Foundation
Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPEAPEAPE) chemistries exploit small electrophilic reagents that react with 2'-hydroxyl groups to interrogate RNARNARNA structure at single-nucleotide resolution. Mutational profiling (MaP) identifies modified residues by using reverse transcriptase to misread a SHAPEAPEAPE-modified nucleotide and then counting the resulting mutations by massively parallel sequencing. The SHAPEAPEAPE-MaP approach measures the structure of large and transcriptome-wide systems as accurately as can be done for simple model RNARNARNAs. This protocol describes the experimental steps, implemented over 3 d, that are required to perform SHAPEAPEAPE probing and to construct multiplexed SHAPEAPEAPE-MaP libraries suitable for deep sequencing. Automated processing of MaP sequencing data is accomplished using two software packages. ShapeMapper converts raw sequencing files into mutational profiles, creates SHAPEAPEAPE reactivity plots and provides useful troubleshooting information. SuperFold uses these data to model RNARNARNA secondary structures, identify regions with well-defined structures and visualize probable and alternative helices, often in under 1 d. SHAPEAPEAPE-MaP can be used to make nucleotide-resolution biophysical measurements of individual RNARNARNA motifs, rare components of complex RNARNARNA ensembles and entire transcriptomes.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据