Cotranscriptional RNA Chemical Probing

Cotranscriptional folding is a fundamental step in RNA biogenesis and the basis for many RNA-mediated gene regulation systems. Understanding how RNA folds as it is synthesized requires experimental methods that can systematically identify intermediate RNA structures that form during transcription. Cotranscriptional RNA chemical probing experiments achieve this by applying high-throughput RNA structure probing to an in vitro transcribed array of cotranscriptionally folded intermediate transcripts. In this chapter, we present guidelines and procedures for integrating single-round in vitro transcription using E. coli RNA polymerase with high-throughput RNA chemical probing workflows. We provide an overview of key concepts including DNA template design, transcription roadblocking strategies, single-round in vitro transcription with E. coli RNA polymerase, and RNA chemical probing and describe procedures for DNA template preparation, cotranscriptional RNA chemical probing, RNA purification, and 3′ adapter ligation. The end result of these procedures is a purified RNA library that can be prepared for Illumina sequencing using established high-throughput RNA structure probing library construction strategies.