Single-cell RNA-seq using UltraMarathonRT expands theknown transcriptome
The ability to map messenger RNA (mRNA) molecules from individual cells using next-generation sequencing technologies, known as single-cell RNA-seq (scRNA-seq), is transforming biology by redefining cellular identities with unmatched detail. However, all current protocols depend on copying RNA into complementary DNA with a single reverse transcriptase (RT) derived from murine leukemia virus, which is an RT enzyme known for low processivity and limited ability to unfold complex RNA structures. Here, for the first time, we introduce a group II intron reverse transcriptase, UltraMarathonRT (uMRT), to perform scRNA-seq. We demonstrate that this enzyme reveals an unexpected transcriptomics landscape by capturing additional genes and other genomic features that conventional RTs miss. We also combined uMRT with metabolic RNA labeling, nucleoside conversion and scRNA-seq to explore genome-wide transcriptome dynamics at the single-cell level. Overall, we establish uMRT as a transformative biotechnological tool for single-cell transcriptomics.