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

The quality of standard single-cell experiments often depends on the immediate processing of cells or tissues post-harvest to preserve fragile and vulnerable cell populations, unless the samples are adequately fixed and stored. Despite the recent rise in popularity of probe-based and aldehyde-fixed RNA assays, these methods face limitations in species and target availability and are not suitable for immunoprofiling or assessing chromatin accessibility. Recently, a reversible fixation strategy known as FixNCut has been successfully deployed to separate sampling from downstream applications in a reproducible and robust manner, avoiding stress or necrosis-related artifacts. In this article, we present an optimized and robust practical guide to the FixNCut protocol to aid the end-to-end adaptation of this versatile method. This protocol not only decouples tissue or cell harvesting from single-cell assays but also enables a flexible and decentralized workflow that unlocks the potential for single-cell analysis as well as unconventional study designs that were previously considered unfeasible. Key features • Reversible fixation: Preserves cellular and molecular structures with the option to later reverse the fixation for downstream applications, maintaining cell integrity • Compatibility with single-cell assays: Supports single-cell genomic assays such as scRNA-seq and ATAC-seq, essential for high-resolution analysis of cell function and gene expression • Flexibility in sample handling: Allows immediate fixation post-collection, decoupling sample processing from analysis, beneficial in settings where immediate processing is impractical • Preservation of RNA and DNA integrity: Effectively preserves RNA and DNA, reducing degradation to ensure accurate transcriptomic and genomic profiling • Suitability for various biological samples: Applicable to a wide range of biological samples, including tissues and cell suspensions, whether freshly isolated or post-dissociated • Enables multi-center studies: Facilitates collaborative research across multiple centers by allowing sample fixation at the point of collection, enhancing research scale and diversity • Avoidance of artifacts: Minimizes stress or necrosis-related artifacts, preserving the natural cellular physiology for accurate genomic and transcriptomic analysis.