Single-cell RNA sequencing (scRNA-seq) has become an essential tool for delineating cellular diversity in normal tissues and alterations in disease states. This technique requires the dissociation of tissue specimens into cell suspensions. However, the isolation of intact cells can be challenging due to factors such as fragility, large size and tight interconnections. Additionally, single-nuclei isolation can be performed on frozen tissue, enabling the analysis of biobanked samples in a single batch. This protocol for single-nuclei RNA sequencing (snRNA-seq) provides an alternative approach to scRNA-seq, overcoming these limitations to generate high-quality transcriptomic data.

The analysis of gene expression at the cellular level has proven to be a powerful tool for understanding various aspects of lung biology and disease, particularly the process of lung aging. Aging affects different cell types within the human lung heterogeneously, leading to a range of associated changes in their function. Examining the patterns of gene expression in the numerous lung cell types provides insights into the aging and disease processes that contribute to cellular dysfunction. By analyzing these changes at the single-cell level, we can delineate the complex cellular diversity in the human lung and track alterations in molecular pathways involved in the dynamic process of lung aging. Understanding these gene expression patterns will offer opportunities for timely interventions and the identification of biomarker for early prognosis and personalized treatment therapies.

This protocol collection describes the process of single nuclei RNA sequencing from nuclei isolation from frozen tissue (whole, or from PCLS), to barcoding, library construction and sequencing.

snRNAseq_ProtocolsIO format_NCE202405.docx