A mannitol-based buffer improves single-cell RNA sequencing of high-salt marine cells

Single-cell RNA sequencing (scRNA-seq) enables discovery of novel cell states by transcriptomic profiling with minimal prior knowledge, making it useful for studying non-model organisms. For most marine organisms, however, cells are viable at a higher salinity than is compatible with scRNA-seq, impacting data quality and cell representation. We show that a low-salinity phosphate buffer supplemented with D-mannitol (PBS-M) enables higher-quality scRNA-seq of blood cells from the tunicate Ciona robusta. Using PBS-M reduces cell death and ambient mRNA, revealing cell states not otherwise detected. This simple protocol modification could enable or improve scRNA-seq for the majority of marine organisms. Overall design: Circulating cells were collected from adult Ciona robusta animals, then run directly analyzed with scRNA-seq. One sample was prepared in high-salt buffer (Ca- and Mg-free buffered artificial seawater), then diluted shortly before scRNA-seq encapsulation with water (libarary name Cr_blood_asw). The other sample was prepared in a low-salt, high osmolarity buffer (0.7 M D-mannitol in PBS) (library name Cr_blood_mannitol).