Single-cell sequencing is a powerful tool for studying cellular heterogeneity, and using isolated nuclei for this process offers several advantages such as minimized dissociation bias, suitability with frozen or FFPE samples, and avoidance of stress responses from dissociation.1,2 The success of this methodology is dependent on efficient isolations and accurate counting of nuclei. This article provides valuable insights and recommendations for optimizing nuclei extraction and counting, enabling important downstream applications including single-nucleus RNA sequencing (snRNA-seq) and assay for transposase-accessible chromatin sequencing (snATAC-seq).
After preparing the desired tissue—whether it's fresh, frozen, or fixed—the next step is the critical task of isolating nuclei. The isolation process requires a specific extraction buffer designed to separate the nuclei without disrupting them. There are several commercially available buffers, but some researchers opt for a custom-formulated buffer. Given the fragility of nuclei and their vulnerability to osmotic lysis, there are several techniques that can enhance the extraction process.
Tip 1: Prepare all materials in advance
For a smoother extraction workflow, have all the materials needed for the protocol set up before starting. This includes preparing the necessary buffers and ensuring the instruments are set according to the protocols. Additionally, buffers should be adjusted to the appropriate temperature to eliminate unnecessary waiting periods.
Tip 2: Maintain cold samples
Keep the samples cold both during and after the extraction. This simple precaution prevents nuclei lysis and can increase the efficiency of the experiment.
Tip 3: Time is of the essence
Work quickly and avoid any pauses in the protocol. Delays can adversely affect the results of the extraction. As previously emphasized, pre-arranging the necessary materials allows for a faster and more efficient workflow.
Tip 4: Identify proper clean-ups
The extraction process varies across tissue types, and depending on the tissue, different pore sizes may be required during the isolation step. Select a cleanup method appropriate for the tissue type in order to remove as much cellular debris as possible; the carryover of this debris can cause complications in downstream stages.
Tip 5: Perform a trial run
Since sequencing experiments can run many thousands of dollars, if possible, it is good practice to perform a trial run on your samples and reagents if you are using a new method for the first time. Isolated nuclei can be QC tested using an automated cell counter with fluorescence counting capabilities.
Nuclei counting and viability assays typically involve Trypan Blue or fluorescence-based staining techniques. Trypan Blue facilitates the counting process by selectively coloring cells with compromised membranes (including the nuclei), giving them a distinct darkened appearance. Although traditionally considered a staple method, Trypan blue staining has its drawbacks. Over time, Trypan blue can crystallize, introducing debris that complicates the counting process. Cellular debris may also take on a dark appearance, making it challenging for automated and manual counting methods to distinguish between debris and dead cells.
Fluorescence-based assays, particularly Acridine orange /Propidium iodide (AO/PI), are the recommended choice as they provide a more objective and accurate count. During this process, AO enters live and unlysed cells, emitting a green fluorescence. In contrast, PI permeates dead or compromised cells and produces a red signal that labels isolated nuclei. This method preserves clarity and avoids any crosstalk as the PI signal absorbs the AO signal due to Förster resonance energy transfer (FRET). Furthermore, each dye only becomes fluorescent upon binding to nucleic acid, which prevents background fluorescence and ignores cellular debris to ensure a more accurate nuclei count than Trypan blue techniques.
Caption: Result image of mouse kidney nuclei. Isolated nuclei appear red, intact cells green, and debris remains unstained. Captured on DeNovix CellDrop Automated Cell Counter.
Both counting processes are further improved through the use of automated cell counters. They enhance the precision and consistency of cell counting over manual methods by eliminating user bias and adopting standardized procedures. Key features of these instruments include low volume requirements for analysis and slide-free operation, which conserves scarce samples and supports environmentally friendly practices. Automated systems also speed up the workflow and allow users to efficiently process a higher throughput of samples to provide more data for robust analysis.
Once the nuclei are successfully isolated, they need to be counted and quality-checked before proceeding to the library preparations. Precise counting begins with the preparation of the reagents AO and PI, collectively referred to as AO/PI.
Tip 1: Equilibrate AO/PI to room temperature
One of the foremost steps in this process is to ensure that the AO/PI solution is equilibrated to room temperature before use. Room temperature AO/PI solution will exhibit the highest fluorescence yield, ensuring optimum efficiency of the assay.
Tip 2: Optimize nuclei counting volume
The counting process involves staining and counting a subset of the isolated nuclei, which, once stained, are unsuitable for the downstream experiment. Due to the small quantity of samples obtained from these extraction methods, conserving nuclei by using only what is necessary for quantification is essential. Fortunately, advanced counting systems allow for the use of minimal input volumes. This allows users to preserve the majority of the sample for the intended experimental procedures.
Tip 3: Gently resuspend delicate nuclei
After adding the staining solution, homogenously resuspended the nuclei before loading. However, rigorous vortexing and pipetting will disrupt the nuclei and lead to sample loss. Considering their fragility, either light pipetting or gentle mixing is the best option for resuspension.
Tip 4: Minimize delay after mixing
Reduce interruptions in the protocol by continuing to move quickly after the nuclei are combined with AO/PI. Prolonged exposure or delays can adversely affect the nuclei, leading to increased osmotic lysis and lower efficiency of the downstream workflow.
Caption: Once nuclei suspension is thoroughly mixed with AO/PI, the sample can undergo a QC check on an automated cell counter.
The presence of debris during cell counting can vary based on the extraction process and the nature of the sample. Although sophisticated cell counting technologies are capable of accurate identification in the presence of debris, minimizing this debris is better for downstream applications.
Carryover debris from extractions poses several challenges. Not only can it obstruct microfluidic systems, but it can also occupy wells intended for nuclei, leading to wasted resources. In addition, difficult samples like those from the brain and heart require thorough nuclei purification because they inherently contain more debris compared to other sample types.
In instances where significant cellular debris is detected during the counting stage, the best course of action is to utilize nuclei clean-ups. A popular approach involves kits equipped with magnetic microbeads. These microbeads bind to the nuclei and facilitate the removal of debris and live cells, which efficiently purifies samples without compromising yields.
Caption: Left–Isolated nuclei from mouse brain tissue. Right–Same sample after purification with Anti-Nucleus Microbeads (Miltenyi Biotec).
Accurate nuclei isolation, counting and QC are vital for reliable downstream applications like single-cell sequencing. Following these best practices enables scientists to refine their nuclei protocols, leading to improved outcomes and more meaningful scientific discoveries.
For scientists aiming to optimize their single-cell genomics workflows, the CellDrop Automated Cell Counter is an excellent choice and eliminates the need for disposable plastic slides. DeNovix CellDrop Series instruments are standalone with intuitive software that provides dedicated applications for Isolated Nuclei QC, primary cells and cell cultures. CellDrop simplifies the process of automatic cell counting and viability assessments, ensuring efficient and accurate results. To learn more about the CellDrop and how it can benefit your single-cell research, visit DeNovix.com or see how it is used in the nuclei counting workflow in this webinar.
1. Habib, N., Avraham-Davidi, I., Basu, A., Burks, T., Shekhar, K., Hofree, M., Choudhury, S. R., Aguet, F., Gelfand, E., Ardlie, K., Weitz, D. A., Rozenblatt-Rosen, O., Zhang, F., & Regev, A. (2017). Massively parallel single-nucleus RNA-seq with DroNc-seq. Nature methods, 14(10), 955–958. https://doi.org/10.1038/nmeth.4407
2. Gaublomme, J. T., Li, B., McCabe, C., Knecht, A., Yang, Y., Drokhlyansky, E., Van Wittenberghe, N., Waldman, J., Dionne, D., Nguyen, L., De Jager, P. L., Yeung, B., Zhao, X., Habib, N., Rozenblatt-Rosen, O., & Regev, A. (2019). Nuclei multiplexing with barcoded antibodies for single-nucleus genomics. Nature communications, 10(1), 2907. https://doi.org/10.1038/s41467-019-10756-2