A practical solution for preserving single cells for RNA sequencing.

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• Single-cell experiments typically require fresh samples, limiting experimental design and accessibility.
• Logistical constraints such as sample transport and equipment scheduling often hinder single-cell studies.

Purpose of the Study:

• To adapt dithio-bis(succinimidyl propionate) (DSP) fixation for stabilizing cells for single-cell transcriptomic analysis.
• To evaluate the impact of DSP fixation on RNA integrity, yield, and data quality in single-cell RNA sequencing.

Main Methods:

• Histological tissue fixation using dithio-bis(succinimidyl propionate) (DSP) was adapted for cell sample stabilization.
• Single-cell RNA sequencing was performed on DSP-fixed and fresh cells to compare data quality.
• Cell viability was assessed using propidium iodide staining in DSP-fixed cells.

Main Results:

• DSP fixation preserves RNA integrity and yield at the gene level, comparable to fresh samples.
• DSP-fixed cells exhibit characteristic RNA-seq artefacts, including reduced cDNA yield and 3' bias.
• Instantaneous fixation with DSP can reduce inter-cell technical variability and allows for viability staining.

Conclusions:

• DSP fixation offers a viable method to stabilize cells for single-cell transcriptomics, overcoming logistical barriers.
• While minor artefacts exist, DSP fixation maintains RNA complexity and reduces technical variability.
• This approach enhances the flexibility and accessibility of single-cell experiments by enabling sample preservation and transport.