PBMC fixation and processing for Chromium single-cell RNA sequencing.

Area of Science:

• Single-cell genomics
• Molecular biology
• Cellular analysis

Background:

• Single-cell transcriptomic analysis is increasingly important for rare cell populations.
• Live cell preparation for single-cell sequencing causes stress and limits experimental design.
• Existing fixation methods are often incompatible with primary cells like immune cells.

Purpose of the Study:

• To develop a methanol-fixation and processing method for preserving human peripheral blood mononuclear cells (PBMCs) for single-cell RNA sequencing (scRNA-Seq).
• To optimize the fixation and rehydration steps to maintain RNA integrity and compatibility with the 10× Chromium platform.

Main Methods:

• Developed a three-step methanol fixation protocol (fixation, storage, rehydration) for PBMCs.
• Investigated RNA degradation during different protocol steps and tested alternative buffers (PBS vs. SSC).
• Validated the optimized protocol using the 10× Chromium scRNA-Seq workflow.

Main Results:

• PBMC RNA degradation occurred during PBS rehydration, not fixation or storage (up to 3 months).
• Resuspension in 3× saline sodium citrate (SSC) buffer, instead of PBS, preserved RNA integrity and prevented leakage.
• Methanol-fixed PBMCs in SSC integrated seamlessly into scRNA-Seq workflows, yielding high-quality data comparable to live cells without altering transcriptional profiles or cell subpopulations.

Conclusions:

• The optimized methanol fixation and SSC buffer protocol effectively preserves primary cell RNA for scRNA-Seq.
• This method enables complex experimental designs, including batching, with fixed primary cells and tissues.
• The protocol is adaptable to various cell types, expanding the utility of single-cell analysis.