Current single cell RNA sequencing (scRNA-seq) technologies rely on the capture of mRNA ends or computational inference of full-length mRNA through transcript-wide fragments (Smart-seq) to study gene expression inside a cell. While attempts are out there to overcome the challenge of full-length RNA capture major issues remain because of e.g. secondary structures or natural RNA modifications. Here, we aim to establish a new single-cell RNA-seq protocol amenable of capturing unforeseen RNA modalities.

RNA-seq approaches rely on the reverse transcription of RNAs molecules into more stable cDNA that can introduce significant biases in RNA-seq readout. The most commonly used RTs are derived from Moloney Murine Leukemia Virus (MMLV) and are typically fast in relay, do multiple cycle reactions, and thus are prone to stop at secondary structures commonly found in long transcripts. In contrast to classical retroviral RTs, Marathon RT, encoded within group II introns of Eubacterium rectale is ultra-processive and can reverse transcribe long transcripts (up to 30 kb) from one end to another in a single run, even in presence of highly stable structural elements. Here, we implemented a scRNA-seq method to capture full-length and structured transcripts using Marathon RT. First, we determined Marathon RTs sensitivity level using synthetic reference transcripts and adapted it to low RNA input. Once the suitability of Marathon RT for single cell level reverse transcription is validated, we incorporated structural probing into the protocol. In an infection setting, this new method would allow us to study not only pathogenic transcripts but also host RNAs in full-length on a single cell level.