Single-cell RNA sequencing (scRNA-seq) has led to an unprecedented understanding of gene expression and regulation in individual cells. Many scRNA-seq approaches rely upon the template switching property of Moloney murine leukemia virus (MMLV)-type reverse transcriptases. Template switching is believed to happen in a sequential process involving non-templated addition of three protruding nucleotides (+CCC) to the 3’ end of the nascent cDNA, which can anneal to the matching rGrGrG 3’ end of the template switching oligo (TSO), allowing the reverse transcriptase (RT) to switch templates and continue copying the TSO sequence.  In this study, we present a detailed analysis of template switching biases with respect to the RNA template, specifically the sequence and nature of its 5’ end (capped vs. non-capped). In addition, we profiled the composition of the non-templated addition in the absence of TSO and demonstrated that the 5’ end of RNA template influences the terminal transferase activity of the RT. Furthermore, we found that designing new TSOs to pair with the most common non-templated additions did little to improve template switching efficiency. Our data provide evidence suggesting that non-templated addition and template switching are concurrent and competing processes, in contrast to the current understanding of the template switching process.<br/>

New insights into the interplay between non-templated addition and template switching by MMLV-based reverse transcriptases

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.