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

Area of Science:

• Marine biology
• Genomics
• Cell biology

Background:

• Single-cell RNA sequencing (scRNA-seq) is a powerful tool for discovering novel cell states.
• Marine organisms present unique challenges for scRNA-seq due to salinity incompatibility.
• Existing methods can lead to poor data quality and limited cell representation in marine species.

Purpose of the Study:

• To develop a protocol for high-quality scRNA-seq in marine organisms.
• To address challenges posed by high salinity environments on cell viability.
• To improve cell representation and data quality for transcriptomic profiling.

Main Methods:

• Developed a low-salinity phosphate buffer supplemented with D-mannitol (PBS-M).
• Applied PBS-M to blood cells from the tunicate Ciona robusta for scRNA-seq.
• Validated the PBS-M protocol in a second tunicate species.

Main Results:

• PBS-M significantly improved scRNA-seq data quality in tunicates.
• The buffer reduced cell death and minimized ambient mRNA contamination.
• Novel cell states, previously undetectable, were revealed using PBS-M.
• Protocol effectiveness was confirmed in a second marine tunicate species.

Conclusions:

• PBS-M is an effective simple modification for enhancing scRNA-seq in marine organisms.
• This protocol can overcome salinity-related barriers in marine single-cell genomics.
• The method has broad potential for advancing research in diverse marine species.