A user guide to RT-based mapping of RNA modifications

Chemical modifications to RNA nucleotides are both a naturally occurring layer of biological regulation and an increasingly prevalent approach to synthetically alter RNA function in therapeutic applications. Detection of their presence, prevalence, and stoichiometry across different RNAs is critical to understanding their underlying functions. However, this remains challenging due to the technical barriers involved in differentiating chemically similar modification species, and in detecting rare or low stoichiometry modifications. Reverse transcription-based techniques rely on the introduction of a predictable mutation, truncation, or deletion signature when a reverse transcriptase encounters a modified nucleotide of interest. Previous studies have shown promise in detecting modifications to single nucleotide resolution, but the low efficiency and processivity of many commercially available reverse transcriptases has resulted in discordant conclusions in some cases. Here, we present guidelines and best practices for applying the highly processive MarathonRT enzyme to reverse transcription-based modification sequencing. These guidelines include recommendations for controls and example protocols to help users plan robust experiments for mapping modification(s) of choice, as well as discussion of the limitations for the methods described.