Figure 1. SHAPE-Seq workflow.

in vitro RNA structures are analyzed by first purifying RNAs of interest, refolding in an appropriate buffer with optional ligands, and modifying with a SHAPE reagent (+) or a control solvent (−). In-cell probing experiments modify RNAs within the cell after the SHAPE reagent or control solvent is added to the media. RT is initiated in one of two ways. In SHAPE-Seq v2.1, an RNA linker sequence is ligated to RNAs that serves as an RT priming site, whereas in-cell probed RNAs are extracted then primed directly with an internal RT priming site, such as within the intrinsic terminator shown. Insets show specific 5′ and 3′ cDNA sequences, the latter of which is used to identify the SHAPE modification position. After RT, all SHAPE-Seq steps are similar. After RNA hydrolysis, a DNA adapter required for Illumina sequencing is ligated to the 3′ cDNA ends. Selective PCR is then used to generate quality assessment or sequencing libraries. The selective PCR uses a selection primer (black) designed to bridge the RT priming site and the 5′ end of the extended cDNA. This allows efficient PCR amplification only if the RT reaction created cDNA extensions. If no cDNA was synthesized, the selection primer cannot bind properly to the RT primer-adapter side product junction (inset), limiting amplification. Quality assessment libraries use a reverse primer that is fluorescently labeled for analysis with capillary electrophoresis, while sequencing libraries use the full Illumina adapter sequence. Subsequent sequencing and bioinformatic analysis (Spats) of the (+) and (−) libraries generates the characteristic SHAPE-Seq reactivity spectra.