Single-cell RNA-seq using UltraMarathonRT expands the known transcriptome.

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• Single-cell RNA sequencing (scRNA-seq) is revolutionizing biology by detailing cellular identities.
• Current scRNA-seq methods use reverse transcriptase (RT) enzymes with limitations in processivity and RNA structure unfolding.
• Murine leukemia virus-derived RT is the standard but misses complex RNA structures.

Purpose of the Study:

• To introduce a novel reverse transcriptase for scRNA-seq applications.
• To evaluate the performance of a group II intron reverse transcriptase, UltraMarathonRT (uMRT), in scRNA-seq.
• To explore enhanced transcriptomic analysis using uMRT.

Main Methods:

• Developed and implemented UltraMarathonRT (uMRT), a group II intron reverse transcriptase, for scRNA-seq.
• Compared uMRT performance against conventional RTs in capturing RNA molecules.
• Integrated uMRT with metabolic RNA labeling and nucleoside conversion for dynamic transcriptome analysis.

Main Results:

• uMRT captured a broader range of genes and genomic features compared to conventional RTs.
• The enzyme revealed an expanded transcriptomics landscape previously inaccessible.
• Combined methods enabled exploration of genome-wide transcriptome dynamics at the single-cell level.

Conclusions:

• UltraMarathonRT (uMRT) significantly advances scRNA-seq capabilities.
• uMRT overcomes limitations of conventional RTs, providing a more comprehensive view of the transcriptome.
• This novel enzyme is a transformative tool for single-cell transcriptomics research.