Single Nuclei Isolation Protocols for snRNA-Seq Analysis

The nucleus is the largest organelle in eukaryotic cells and houses the DNA within a double membrane nuclear envelope that organizes chromatin for efficient packing and gene regulation, which profoundly influences the cell's epigenetic and transcriptomic profiles. In the last decade, the use of isolated nuclei for molecular studies, particularly in genomics, transcriptomics, and epigenomics, has increased significantly, driven by advancements in high-throughput sequencing technologies and declining sequencing costs, but above all, the rise of single cell sequencing technologies [1]. Studying the transcriptomic profiles of single nuclei is crucial in many applications aiming to resolve the complexities of gene expression and cellular diversity, especially when single whole cells transcriptomics is not readily accessible. Optimizing the techniques and protocols that allow for best single nuclei isolation is fundamental for downstream advanced genomic studies, such as single-nucleus RNA sequencing, though each method presents its own challenges and limitations. This review aims to examine various protocols for nuclei isolation from diverse sources, including from human umbilical cord blood (CD34+ cells) [5], kidney tissue [3], organoids [8], mouse brain [7], heart tissue [6], and plant tissue [2]. Each method is assessed for its efficiency with a comparative analysis of mechanical and chemical dissociation approaches. Furthermore, the methods’ yield, and quality are assessed with particular consideration for implications in downstream bioinformatics analyzes and workflows [4]. Our findings offer a comprehensive evaluation of nuclei isolation techniques and single nuclei bioinformatics analyzes, enabling the upstream optimization for specific tissue types and research applications.