High-resolution single-cell sequencing of trans-spliced mRNA

Single-cell RNA sequencing (scRNA-seq) has revolutionized transcriptomic analyses by enabling the study of cellular heterogeneity at unprecedented resolution. However, most existing scRNA-seq methods have limited sensitivity, particularly in non-mammalian systems, which typically contain far less RNA per cell than mammalian cells. Current approaches to sequencing RNAs in non-mammalian cells rely on inherently inefficient and often non-specific molecular strategies, such as template-switching or C-tailing, to add a common sequence to the 5′ end of cDNAs for downstream amplification. These steps substantially limit both the sensitivity and specificity of transcript detection, particularly in cells with low mRNA content. To overcome these limitations, we developed SL-Smart-seq3xpress, a high-sensitivity scRNA-seq protocol optimized for organisms that perform trans-splicing. These include kinetoplastids, marine dinoflagellates and nematodes, such as Caenorhabditis elegans, which add a conserved spliced leader (SL) sequence to the 5′ end of nearly all mRNAs. By directly targeting this SL sequence using a specific primer, SL-Smart-seq3xpress bypasses the need for inefficient 5′ sequence-adding steps and captures >80% of mRNA molecules in individual Trypanosoma brucei cells, substantially more than all other techniques to date. From single-cell sorting to libraries ready for sequencing, the protocol can be completed in 3 d by an experienced technician or graduate-level molecular biologist. The resulting increase in sensitivity, coupled with the compact genomes of these organisms, enables not only high-resolution transcriptomic profiling but also inference of genome-level structural variation at the single-cell level from mRNA data alone. We have successfully applied this method to study transcriptional and genomic heterogeneity in T. brucei and expect it to be useful for other SL-containing eukaryotes. SL‑Smart‑seq3xpress is a highly sensitive single-cell RNA sequencing method that is compatible with organisms that undergo trans‑splicing and enables high‑resolution analysis of transcriptional and genomic heterogeneity in single cells.