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

Area of Science:

• Molecular Biology
• Neuroscience
• Genomics

Background:

• Single-nucleus RNA sequencing (snRNA-seq) is vital for studying complex tissues, but its success hinges on nuclear isolation methods.
• Existing techniques for isolating nuclei vary in preserving nuclear integrity and minimizing contamination, with a lack of systematic comparison for brain tissue.

Purpose of the Study:

• To systematically evaluate and compare three distinct nuclei isolation methods for snRNA-seq.
• To assess the impact of different isolation strategies on nuclear integrity, RNA yield, purity, and downstream data quality.
• To guide researchers in selecting optimal protocols for brain tissue snRNA-seq experiments.

Main Methods:

• Comparison of three mechanistically distinct nuclei isolation strategies: sucrose gradient centrifugation, spin column-based method, and a machine-assisted platform.
• Evaluation of nuclei isolation methods using brain tissues.
• Assessment of cell type proportions, transcriptional homogeneity, marker preservation, and RNA contamination levels.

Main Results:

• All tested methods successfully captured diverse cell types in brain tissue.
• Significant protocol-dependent differences were observed in cell type proportions and transcriptional homogeneity.
• Isolation workflows differentially impacted the preservation of cell-type-specific markers and contamination levels (ambient, mitochondrial, ribosomal RNA).

Conclusions:

• Nuclei isolation methodology is a critical experimental variable that profoundly influences snRNA-seq data quality.
• The choice of isolation method significantly shapes biological interpretation of snRNA-seq data.
• This study provides crucial insights for optimizing snRNA-seq protocols in brain research.