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

## TL;DR

This study compares three methods for isolating nuclei from brain tissue for RNA sequencing, finding that the machine-assisted method provides the best results with minimal contamination and consistent data.

## Contribution

The study provides the first systematic comparison of three nuclei isolation methods for brain snRNA-seq, revealing significant differences in data quality and cell-type representation.

## Key findings

- Isolation protocol affects brain cell type proportions and transcriptional homogeneity.
- Machine-assisted isolation yields the most intact nuclei with minimal contamination.
- Glial and neuronal gene expression patterns differ among isolation protocols.

## Abstract

Single-nucleus RNA sequencing (snRNA-seq) enables resolving cellular heterogeneity in complex tissues by using nuclei instead of cells, overcoming limitations of single-cell RNA sequencing and enabling analysis of frozen and hard-to-isolate tissues. Despite advances in isolation techniques, systematic evaluations of their effects on nuclear integrity and subsequent data quality remain lacking, a critical gap with profound implications for rigor and reproducibility. To address this, we compared three mechanistically distinct nuclei isolation strategies with brain tissue: a sucrose gradient centrifugation-based method, a spin column-based method, and a machine-assisted platform. All methods captured diverse cell types but revealed considerable protocol-dependent differences in cell type proportions, transcriptional homogeneity, and the preservation of cell-state-specific markers. Moreover, workflows differentially influenced contamination levels from ambient, mitochondrial, and ribosomal RNAs, with the machine-assisted method exhibiting the highest overall data quality. Our findings establish nuclei isolation methodology as a critical experimental variable shaping snRNA-seq data quality and biological interpretation.

•Comparison of three nuclei isolation methods for brain single-nucleus RNA sequencing•Isolation protocol affects brain cell type proportions and transcriptional homogeneity•Glial and neuronal gene expression patterns differ among isolation protocols•Machine-assisted isolation yields the most intact nuclei with minimal contamination

Comparison of three nuclei isolation methods for brain single-nucleus RNA sequencing

Isolation protocol affects brain cell type proportions and transcriptional homogeneity

Glial and neuronal gene expression patterns differ among isolation protocols

Machine-assisted isolation yields the most intact nuclei with minimal contamination

Existing nuclei isolation techniques for snRNA-seq vary in their ability to preserve nuclear integrity, minimize ambient RNA contamination, and optimize recovery rates. This poses a challenge for researchers in choosing the most suitable approach for their particular experimental requirements. To address this critical issue, our study directly compared three nuclei isolation methods and evaluated their performance in terms of yield, purity, and downstream sequencing quality. By providing a comprehensive assessment, we aim to guide researchers in selecting the most appropriate isolation protocol for their snRNA-seq experiments, ensuring optimal results and advancing the study of complex brain tissues at the single-nucleus level.

Kersey et al. systematically evaluate three nuclei isolation methods for brain snRNA-seq, demonstrating that protocol choice markedly affects data quality metrics, including nuclei yield and ambient RNA contamination levels, as well as cell type proportions. Notably, a machine-assisted approach minimizes technical variability, providing consistent transcriptional signatures across glial and neuronal populations.