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In Vivo Genome-Wide RNA Structure Probing with Structure-seq
Laura E Ritchey1,2,3, Zhao Su4, Sarah M Assmann2,4
1Department of Chemistry, Pennsylvania State University, University Park, PA, USA.
Methods in Molecular Biology (Clifton, N.J.)
|April 5, 2019
Summary
This chapter details the Structure-seq2 method for in vivo RNA structure probing, offering high-quality transcriptome-wide data. It emphasizes controls and troubleshooting for accurate analysis of cellular RNA structures.
Area of Science:
- Molecular Biology
- Genomics
- Biochemistry
Background:
- In vivo RNA structure probing offers a global view of cellular RNA architecture.
- Understanding RNA structure is crucial for elucidating its diverse biological functions.
- Existing methods may require technical precision, necessitating detailed protocols.
Purpose of the Study:
- To present a detailed protocol for generating high-quality transcriptome-wide RNA structure data using Structure-seq2.
- To focus on essential controls and troubleshooting for the Structure-seq2 method.
- To enable the application of Structure-seq2 across various organisms and chemical reagents.
Main Methods:
- Structure-seq2 protocol for in vivo genome-wide RNA structure probing.
- Detailed steps focusing on technical precision, controls, and troubleshooting.
- Application of various RNA-modifying chemicals (DMS, glyoxal, SHAPE reagents).
Main Results:
- High-quality transcriptome-wide RNA structure data in vivo.
- Demonstration of Structure-seq2 applicability in plants, humans, and bacteria.
- Compatibility with multiple RNA-modifying chemicals.
Conclusions:
- Structure-seq2 provides a robust method for in vivo RNA structure analysis.
- The protocol emphasizes critical steps for reliable data generation.
- Generated data is amenable to analysis with the StructureFold2 computational pipeline.
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