Comparative analysis of nuclei isolation methods for brain single-nucleus RNA sequencing.

Area of Science:

• Neuroscience
• Genomics
• Molecular Biology

Background:

• Single-nucleus RNA sequencing (snRNA-seq) is vital for analyzing complex tissues, especially frozen or difficult-to-isolate samples.
• Existing snRNA-seq protocols lack systematic evaluations of nuclei isolation method impacts on data quality and reproducibility.

Purpose of the Study:

• To compare the effects of three distinct nuclei isolation strategies on snRNA-seq data quality.
• To assess the influence of isolation methods on nuclear integrity, cell type proportions, and RNA contamination.

Main Methods:

• Comparison of three nuclei isolation methods: sucrose gradient centrifugation, spin column, and a machine-assisted platform.
• Analysis of brain tissue using snRNA-seq to evaluate cell type diversity, transcriptional homogeneity, and marker preservation.
• Assessment of contamination from ambient, mitochondrial, and ribosomal RNAs.

Main Results:

• All methods identified diverse cell types, but protocol-dependent variations in cell type proportions and marker preservation were observed.
• The machine-assisted method demonstrated superior overall data quality, with lower contamination levels.
• Nuclei isolation methodology significantly impacts transcriptional homogeneity and biological interpretation.

Conclusions:

• Nuclei isolation strategy is a critical variable in snRNA-seq experiments.
• Method choice directly influences the accuracy of cell type identification and downstream biological insights.
• Standardizing or carefully selecting isolation protocols is essential for robust and reproducible snRNA-seq studies.