A modular transcript enrichment strategy for scalable, atlas-aligned, and clonotype-resolved single-cell transcriptomics

Abstract Existing targeted approaches either rely on per-gene barcoded probes and transcript tiling, which limit scalability, or forgo reverse transcription, precluding capture of highly variable transcripts. Here, we present targeted reverse transcription-linker (TRTL), a modular method that can be integrated into existing single-cell transcriptomic workflows to enable targeted readout of user-defined transcript panels, with minimal changes in protocol when scaling from tens to thousands of genes. Because TRTL retains reverse transcription, it also enables the capture of variable transcripts, such as TCR and BCR sequences. Applying TRTL and combinatorial indexing RNA-seq to the mouse brain, we show that carefully designed panels support robust alignment to existing reference atlases, enabling accurate cell type annotation and detection of cellular populations at low sequencing depths. Lastly, we combine TRTL with nuclear hashing-based multiplexing for a targeted-sci-Plex protocol and further demonstrate that targeted-sci-Plex can resolve dynamic T-cell fate trajectories following diverse activating exposures while concurrently profiling T-cell clonotypes.