Knowledge of RNA structure is critical to understanding both the important 
functional roles of RNA in biology and the engineering of RNA to control 
biological systems. This article contains a protocol for selective 2'-hydroxyl 
acylation analyzed by primer extension and sequencing (SHAPE-Seq) that, through 
a combination of structure-dependent chemical probing and next-generation 
sequencing technologies, achieves structural characterization of hundreds of 
RNAs in a single experiment. This protocol is applicable in a variety of 
conditions, and represents an important tool for understanding RNA biology. The 
protocol includes methods for the design and synthesis of RNA mixtures for 
study, and the construction and analysis of structure-dependent sequencing 
libraries that reveal structural information of the RNAs in the mixtures. The 
methods are generally applicable to studying RNA structure and interactions in 
vitro in a variety of conditions, and allows for the rapid characterization of 
RNA structures in a high-throughput manner. Curr. Protoc. Chem. Biol. 4:275-297 
© 2012 by John Wiley & Sons, Inc.