Chemical probing, for decades, has been one of the most popular tools for studying the secondary structure of RNA molecules. Recently, protocols for simultaneous analysis of multiple RNAs have been developed, enabling in vivo transcriptome-wide interrogation of the RNA structure dynamics. One of the most popular methods is the selective 2′-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP). In this study, we describe the evaluation of this protocol by addressing the influence of the reverse transcription enzymes, buffer conditions, and chemical probes on the properties of the cDNA library and the quality of mutational profiling-derived structural signals. Our results reveal a SuperScript IV (SSIV) reverse transcriptase as a more efficient enzyme for mutational profiling of SHAPE adducts and shed new light on the role of Mn2+cations in the modulation of SSIV readthrough efficiency.