In Vivo Genome-Wide RNA Structure Probing with Structure-seq
In vivo genome-wide RNA structure probing provides a global view of RNA structure as it occurs in the cell and can assist in elucidating important functional aspects of RNA structure. Structure-seq2 provides high-quality data on transcriptome-wide RNA structure in vivo but contains numerous steps that require technical precision. In this chapter we present the steps needed to produce high-quality structural data with a focus on controls and troubleshooting. Structure-seq2 can be applied to numerous organisms including plants, humans, and bacteria and is amenable to a wide variety of RNA-modifying chemicals including DMS, glyoxal, and SHAPE reagents. Notably, the data generated by the method highlighted here can be readily analyzed using our StructureFold2 computational pipeline.
Key wordsRNA structure probing Structure-seq Illumina sequencing StructureFold2 DMS Glyoxal SHAPE 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)
We thank Dr. David Mitchell and Dr. Joseph Waldron for helpful input on the manuscript and Dr. Craig Praul (Genomics Core Facility, Penn State University) for high-throughput Illumina sequencing. This work was supported by the National Science Foundation Plant Genome Research Program (NSF-IOS-1339282), with additional support from an Innovation Award from Penn State University.
DMS treatment . Here we show some appropriate safety precautions needed for the DMS reaction. We also demonstrate how to perform the DMS treatment using Arabidopsis as an example (MOV 101517 kb)
PAGE excision . Here we demonstrate how to dice the gel pieces at various steps throughout Structure-seq2 (MOV 19,112 kb)
Streptavidin elution . Here we demonstrate how to elute the biotinylated products from streptavidin magnetic beads (MOV 72697 kb)
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