The described method excludes lengthy procedures for debris removal. Herein, we present an adapted method for single nuclei isolation from small needle biopsies independent of preservation method (e.g., fresh-frozen, flash-frozen, or RNA later-stored). The limitations of the current methods prevent the processing of such tissues, precluding the evaluation of critical clinical samples collected at unexpected times and away from the laboratory. There are also no published methods for obtaining intact single nuclei from RNA later-stored samples, a common approach used for biobanking clinical biopsies. Currently, there are few publications reporting a step-by-step protocol for the application of single nuclei from clinical samples, none of which utilize small needle biopsies 13, 14, 15. Aggregate and debris removal is typically accomplished through lengthy ultra-centrifugation or cell sorting techniques which further decrease nuclei quality and total yield. However, mechanical homogenization leads to over lysis and isolation of unhealthy, ‘blebbing’ nuclei. A common strategy utilizes mechanical homogenization in low concentration detergents to disrupt cellular membranes while releasing intact nuclei, followed by removal of aggregated nuclei and debris 9, 10, 11, 12. Although snRNA-seq is still within its nascent stages, there is a growing number of publications that have been validated for fresh or flash frozen tissue sections and cell lines. Nuclei isolation is not biased towards common cell types and is more suited to identify the cellular etiology of pathology, due to morphological similarities. SnRNA-seq provides an attractive alternative to overcome technical complications introduced during sample preparation. Recently, single nuclei RNA-seq (snRNA-seq) has been introduced as a method to surpass these limitations, whereby cytosolic components are forgone for a clearer snapshot of gene expression 6, 7, 8. Moreover, human tissue biopsies collected for clinical diagnostics are obtained at any time of day and often away from the laboratory, making the fast processing of fresh viable and healthy cells unpractical. This produces a biased representation of cellular architecture 5. In addition, with tissues that have been fixed or partially dissociated, the cell isolation method can preferentially select for easily dissociable cell types. ![]() This process typically relies upon enzymatic incubations at elevated temperatures that can introduce cellular stress artifacts and noise that is only evident after sequencing 3, 4. However, an essential prerequisite for scRNA-seq is the processing of tissues into a homogeneous single cell suspension. Specifically, single cell RNA-sequencing (scRNA-seq) provides novel insight into cellular heterogeneity across human tissues and facilitates the study of complex diseases. The ability to capture gene expression at the single cell resolution has propelled significant discoveries in the biomedical field over the last decade 1, 2. Future applications of this protocol will allow for thorough investigations of small biobank biopsies, identifying cell-specific injury pathways and driving the discovery of novel diagnostics and therapeutic targets. Cellular disassociation did not induce cellular stress responses, which recapitulated transcriptional patterns associated with standardized methods of nuclei isolation. ![]() Quality control measurements demonstrated that these optimizations eliminated cellular debris and allowed for a high yield of high-quality nuclei and RNA for library preparation and sequencing. Using snRNA-seq, 16 distinct kidney cell clusters were recovered from normal and peri-transplant acute kidney injury allograft samples, including immune cell clusters. This method is effective for obtaining normal nuclei morphology without signs of structural damage. Samples can be processed in 90 min or less. The described protocol is fast, low cost, and time effective due to the elimination of cell sorting and ultra-centrifugation. Here, an optimized protocol for single nuclei isolation is presented for frozen and RNA later preserved human kidney biopsies. However, the study of complex tissues using small core biopsies presents many technical challenges. snRNA-seq largely relies on the dissociation of intact nuclei from human tissues. Single cell resolution enables the study of novel cell types, biological processes, cell trajectories, and cell–cell signaling pathways. Single nuclei RNA sequencing (snRNA-seq) has evolved as a powerful tool to study complex human diseases.
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