Single nuclei isolation is a laboratory method for separating a cell’s nucleus from other cellular components. This technique allows researchers to investigate the genetic and epigenetic information within individual cells on a massive scale. By focusing on the nucleus, scientists can obtain a clear snapshot of a cell’s identity and function. This approach is useful for analyzing complex tissues and understanding how individual cells contribute to an organ’s overall function.
Rationale for Isolating Nuclei Over Whole Cells
An advantage of isolating nuclei is the ability to work with frozen tissue samples. This capability expands research by allowing scientists to analyze archived tissues, including clinical samples that are stored frozen. Freezing damages the fragile outer membrane of a whole cell, but the nuclear membrane remains intact, preserving the genetic material. This circumvents the need for fresh tissue, which may not be available.
The technique is also useful for tissues that are difficult to separate into individual, intact cells. Tissues like the brain, heart, and fibrotic tumors have cells that are tightly interconnected or embedded in a dense extracellular matrix. Standard methods to break down these tissues can be harsh, failing to yield viable single cells or losing certain cell types, which introduces bias. Isolating nuclei provides a more reliable alternative for these samples.
Using nuclei also minimizes artificial changes in gene activity caused by the stress of dissociation. The process of separating whole cells can trigger a stress response that alters which genes are turned on or off, obscuring the cell’s true biological state. Because nuclei isolation is a faster and gentler process, the resulting data more accurately reflects the cell’s gene expression profile as it existed within the tissue.
The Isolation Protocol
Tissue Homogenization
The first step is to gently break apart the tissue to free the cells, a process done on ice to prevent degradation. Researchers use a Dounce homogenizer, where a pestle mechanically shears the tissue into a slurry of individual cells and smaller clumps. The amount of force and number of strokes are carefully calibrated to disrupt the tissue structure without damaging the nuclei.
Cell Lysis
Next, the cell membrane is broken open to release the nuclei using a lysis buffer containing a mild detergent. This detergent selectively perforates the outer cell membrane while leaving the nuclear membrane intact. The buffer also contains agents to maintain the proper pH and osmotic pressure, ensuring the nuclei remain stable.
Filtration and Centrifugation
The resulting mixture, or lysate, contains freed nuclei, cytoplasmic debris, and any remaining intact cells. To purify the nuclei, this lysate is passed through a cell strainer to filter out larger debris. The filtered liquid is then centrifuged, causing the denser nuclei to form a pellet at the bottom of the tube while lighter components remain suspended in the liquid above.
Quality Assessment
Before analysis, researchers confirm the quality of the isolated nuclei by examining a small sample under a microscope. They use a DNA-binding dye, like DAPI, which makes the nuclei easy to visualize and count. This step ensures a high concentration of clean nuclei with minimal contamination from debris or unlysed cells, confirming the sample is suitable for analysis.
Downstream Analysis Techniques
Single-Nucleus RNA Sequencing (snRNA-seq)
A common application for isolated nuclei is single-nucleus RNA sequencing (snRNA-seq). This technique measures the gene expression profiles of thousands of individual nuclei simultaneously. Inside each nucleus are messenger RNA (mRNA) molecules, which are copies of genes actively being used by the cell. By sequencing these mRNA molecules, researchers can determine which genes were “on” in each cell, providing a molecular fingerprint that reveals its type and function.
This technology is used to create cellular atlases of complex tissues, such as the human brain, by identifying all the different cell types present. For example, snRNA-seq can be used on postmortem brain tissue from patients with neurological disorders. This reveals cell-type-specific changes in gene expression associated with a disease, helping uncover how specific cells contribute to organ health and pathology.
Single-Nucleus ATAC-seq (snATAC-seq)
Another application is single-nucleus Assay for Transposase-Accessible Chromatin using sequencing (snATAC-seq). This method maps the “open” or accessible regions of chromatin, the complex of DNA and proteins within the nucleus. The accessibility of chromatin is directly related to gene regulation, as open regions of DNA are more likely to be actively transcribed, providing insights into the regulatory landscape of the genome.
This technique reveals which parts of the DNA are available for transcription factors to bind, showing which genes are poised to be turned on or off. When combined with snRNA-seq, it offers a multi-layered view of a cell’s regulatory state. This integrated approach is useful for understanding the gene regulatory networks that define cell identity and drive cellular changes during development and disease.