FixNCut: A Practical Guide to Sample Preservation by Reversible Fixation for Single Cell Assays

Area of Science:

• Single-cell biology
• Molecular biology
• Genomics

Background:

• Standard single-cell experiments require immediate sample processing to maintain cell integrity.
• Existing fixation methods like aldehyde fixation have limitations in species availability and downstream application compatibility.
• Stress and necrosis artifacts can compromise data quality in fragile cell populations.

Purpose of the Study:

• To present an optimized and robust practical guide for the FixNCut reversible fixation protocol.
• To enable flexible, decentralized single-cell analysis workflows by decoupling sample collection from downstream assays.
• To facilitate the adaptation of FixNCut for various single-cell applications and unconventional study designs.

Main Methods:

• Optimization of the FixNCut reversible fixation protocol for broad applicability.
• Demonstration of FixNCut's compatibility with single-cell RNA sequencing (scRNA-seq) and single-cell ATAC sequencing (scATAC-seq).
• Validation of FixNCut's ability to preserve RNA and DNA integrity and minimize cellular artifacts.

Main Results:

• The optimized FixNCut protocol effectively decouples sample harvesting from downstream single-cell assays.
• FixNCut preserves cellular and molecular structures, including RNA and DNA integrity, while minimizing artifacts.
• The protocol supports a flexible workflow, enabling multi-center studies and diverse sample types.

Conclusions:

• The FixNCut protocol offers a versatile solution for preserving sample quality and enabling advanced single-cell analyses.
• This method enhances flexibility in sample handling and processing, overcoming limitations of traditional approaches.
• FixNCut unlocks new possibilities for single-cell research, including large-scale collaborative studies and complex experimental designs.