Single cell sequencing is a cutting-edge technique for analyzing nucleic acids within individual cells from a single tissue source, enabling a granular view of their genetic and epigenetic landscapes within populations of cells. [1]
Among its key applications, scRNA-seq (single cell RNA-seq) can describe transcriptomic profiles and gene expression dynamics, [2] while ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) addresses the accessibility of chromatin, providing insights into regulatory mechanisms. [3]
High-quality nuclei extraction is pivotal for these applications, as the purity and integrity of nuclei directly influence the accuracy and depth of the sequencing data.
This article provides practical tips to prepare high-quality nuclei extracts and tailor your extraction protocol to your sample; explains methods to count nuclei and assess their quality, including their pros and cons; and demonstrates how automated nuclei counting can improve your workflow.
Challenges of Extracting and Counting Nuclei for Single Cell Sequencing
Nuclei extraction involves removing the cell membrane via lysis while keeping the nuclear membrane intact. The resulting debris and exposed, structurally-delicate nuclei pose significant practical challenges, including:
- Sample heterogeneity
- Sample integrity
- Sample contamination
- Nuclei aggregation
- Quantification accuracy
Furthermore, these technical challenges can impact the reliability and scalability of your nuclei extraction.
Practical Tips for Nuclei Extraction
Tailoring your extraction protocol to your specific tissue type will help you overcome the challenges outlined in the previous section. Keep a detailed record of all the different approaches you try to learn what works best for your sample. However, the following practical actions will improve any extraction protocol:
- Prepare all your buffers, dilutions, and equipment beforehand
- Pre-equilibrate them to the temperature outlined in your extraction protocol
- Understand what clean-up your sample will require
- Keep your samples cold to minimize osmotic lysis of the extracted nuclei
- Work quickly and efficiently to further minimize nuclei damage
Section Large Tissue Samples into Smaller Pieces
If you have a large tissue section, consider cutting it up into smaller pieces before you grind it up to help increase the efficiency of nuclei extraction. To ensure the heterogeneity of your nuclei extracts, eliminate as much tissue containing unwanted cell types as possible.
Use an Automated Tissue Dissociator for Consistent Extraction
Grinding your samples using a tissue dissociator rather than manually can help improve your batch-to-batch consistency in your lab and ensure that all your samples are processed the same way.
Be Gentle to Avoid Osmotic Lysis and Mechanical Nuclei Damage
The cell membrane protects the nuclei in regular, healthy cells. However, when you lyse cells for nuclei extraction, the nuclei become exposed to everything previously kept outside the cell. Because the nuclear membrane is more fragile than the cell membrane, the nuclei are susceptible to osmotic lysis. Working quickly and keeping everything cool minimizes this.
They are also extremely fragile and susceptible to mechanical damage, so perform all manual handling of your extracted nuclei gently.
Optimize Filtration to Eliminate Sample Debris
To eliminate as much cellular debris from your nuclei sample as possible before moving to sequencing, aim to learn what specific filtration your sample will require.
The size and abundance of cellular debris in your sample will vary depending on your tissue type and lysis method, so choose the pore size of your filter accordingly. We’ll discuss other steps to eliminate sample debris in a later section.
Economize Your Sample when Performing Quality Control Checks
Because nuclei extraction protocols often yield small sample volumes, which may have only a sparse number of nuclei, aim to minimize the amount you use for nuclei counting and quality control to ensure you have plenty left for sequencing.
Use Fluorescence Rather than Brightfield Methods to Count Nuclei
Although your choice of using fluorescence rather than brightfield methods to count nuclei may be constrained by the instruments you have, fluorescence-based methods offer significant advantages.
Brightfield Methods Using Trypan Blue
Trypan blue is an azo dye that binds to and stains intracellular proteins, and it remains a popular choice to test cell viability. Due largely to its size (873 Da), it cannot pass through the cell membrane and, therefore, stains dead cells, cells with compromised membranes, isolated nuclei, and cell debris.
Under the microscope, live cells will be clear (unstained), while dead and degraded cells, nuclei, and cell debris will be blue/blue-black.
Tips for Using Trypan Blue
To prevent the quality of your trypan blue from complicating your nuclei counting, inspect a few microliters under a microscope.
If you see some crystallites or contaminants in your trypan blue solution, try centrifuging it at a relatively high speed. Isolate the top portion of the solution, filter it, and reinspect it to see if it’s any cleaner. Another option is to run the trypan blue through a 0.2 µm filter to remove the large debris. If neither method works to remove the debris, buy new stocks.
Gently mix your sample and the trypan blue 1:1 in a fresh tube and incubate the mixture for 3–5 minutes, then proceed to nuclei counting. For a detailed explanation of testing cell viability using trypan blue, read this protocol by Warren Strober. [4]
Fluorescence Methods Using Acridine Orange and Propidium Iodide
Acridine Orange and Propidium Iodide (AO/PI) are fluorescent dyes that intercalate nucleic acid.
Acridine Orange is the smaller of the two (265 Da), can cross the cell membranes of living cells, and fluoresces green (at 520 nm). [5]
Propidium Iodide is larger (668 Da) and is excluded from live cells (like trypan blue). It can, however, cross the compromised membranes of dead or dying cells and fluoresces red (at 618 nm). [6]
So, any successfully extracted nuclei will fluoresce red, and any unlysed cells will fluoresce green. This means you get AO and PI in any dead cell/nuclei; however, the green fluorescence from AO is transferred via Förster resonance energy transfer to PI and quenched. This results in a binary situation where:
- Nuclei are red
- Intact cells are green
Because AO and PI bind to nucleic acid, any object in your sample that does not have a nucleus (such as debris or red blood cells) won’t fluoresce. Check out Figure 1 below to see what nuclei samples stained with AO/PI look like.
Compared to trypan blue, using AO/PI provides more accurate estimates of the total number of cells and cell viability, and the results are more objective. Because AO/PI are fluorescent dyes, they have superior detection sensitivity than trypan blue.
Tips for Using AO/PI
Although it’s best to pre-cool your other reagents, leave the AO/PI at room temperature to avoid temperature-induced fluorescence quenching.
The optimal ratio of AO/PI-to-sample is 1:1. Add the two to a fresh tube and gently pipette up and down a few times to mix them thoroughly but remember that the nuclei are extremely fragile. Avoid vortexing if you can.
Note also that AO/PI does not require an incubation time, so you can proceed straight to nuclei counting after mixing. Read this article for a detailed protocol for counting viable cells using AO/PI.
Perform Pre-sequencing Quality Control Checks on Nuclei Samples
Many downstream applications for extracted nuclei are expensive and lengthy. Plus, you get a large amount of complex data, which will be either degraded in quality or hard to interpret if the starting sample is poor. Therefore, spending some on quality control to gauge the suitability of your sample for your intended application is paramount.
Visualize and Quantitate Debris
Debris left over from the nuclei extraction can negatively impact downstream results such as sequencing data, especially if there is a significant amount of it.
Debris can also clog microfluidic devices and take up wells intended for nuclei, leading to wasted time and resources. Therefore, it’s crucial to assess the amount of debris in your sample before continuing to ensure it is fit for purpose.
Check out Figure 2 below to see when the level of debris in your sample is fine and when it’s likely to cause problems in your downstream applications.
If your sample contains lots of debris—all is not lost. You can purify it using Anti-Nucleus MicroBeads from Miltenyi Biotec, which specifically bind to the extracted nuclei to eliminate unwanted debris and live cells without impacting your yield.
Measure Extraction Efficiency
Calculating the extraction efficiency of nuclei lets you assess the effectiveness of your nuclei extraction protocol.
It’s defined as the proportion of nuclei extracted from the cells in a given sample relative to the total number of nuclei present, either extracted or remaining in unlysed cells. You can calculate it by following these steps:
- Determine the total number of cells in your starting sample. You can do this using a hemocytometer or automated cell counter.
- Calculate your expected nuclei count. Assuming each cell contains one nucleus, your expected nuclei count should ideally match the initial cell count.
- Count the number of nuclei obtained after extraction. If you have used fluorescent dyes like AO/PI to label the nuclei, you can count them using fluorescence microscopy or an automated cell counter.
- Calculate the nuclei extraction efficiency using the following formula.
100 x (nuclei per mL) / (nuclei + cells per mL)
What is an Acceptable Nuclei Yeild and Extraction Efficiency?
While low nuclei yields will impact the quality of the data you get from single cell or nuclei sequencing, there is no hard and fast rule regarding what yield to aim for.
However, empirically determining your nuclei yield allows you to quantify an acceptable value suitable for your downstream application.
The same applies to calculating the nuclei extraction efficiency.
If there are 100 events on the screen, but only 50 are nuclei, you have 50 cells that will not get sequenced. An extraction efficiency of 50% probably means you must extend the lysis period or refresh your lysis buffers and try again.
While you should aim for as high extraction efficiency as possible, no target value applies to all applications.
Manufacturers of downstream applications may provide guidance for these quality metrics.
Consider Automated Cell and Nuclei Counting
As mentioned, precise quantification of nuclei is critical for downstream applications. Manual nuclei quantification is slow and prone to user subjectivity.
Automated nuclei quantification using a cell counter is a superior choice for almost all downstream applications because it’s:
- Unbiased
- Standardized
- Scalable
- Fast
- Accurate
To learn more about the benefits and best practices of automated cell counting, download a free copy of Automated Counting of Isolated Nuclei.
Count Cells Without Slides Using CellDrop™
The CellDrop™ Automated Cell Counter offers all the benefits of automation listed above and lets you fine-tune the following event detection criteria:
- Cell diameter and roundness
- Nuclei diameter and roundness
- Fluorescence threshold for each channel
- Irregular cell types/shapes
Plus, it can count cells without disposable slides to reduce the number of consumables you need and provide clear images.
Not only does this reduce the carbon footprint of your lab, keep costs down, and help any green initiatives at your workplace, but it also offers a performance benefit.
The flat sapphire surfaces and lack of requirement for consumables minimize the CellDrop’s™ measurement-to-measurement variability, giving you more reliable counts than instruments that require plastic slides or hemocytometers.
Furthermore, the precision-controlled sample arm allows you to use as little as 2.5 µL of your nuclei extract for counting and quality control, meaning you have more of your sample left for sequencing.
It’s even available in green! Learn More about the CellDrop™.
Optimizing Nuclei Extraction and Counting Summarized
From practical tweaks such as pre-chilling your reagents and working quickly to taking the time to investigate your sample’s clean-up requirements, the tips outlined in this article will help you prepare high-quality nuclei extracts.
Tailoring your nuclei extraction protocol to suit your sample and investing time in quality control will ensure superior single-cell sequencing results.
For further expert advice on optimizing your nuclei extraction and counting, check out our webinar with Application Scientist Ben Capozzoli.
FAQs
Q: How can I avoid nuclei clumping in my extracts?
A: First, ensure that your cells are healthy when you begin to extract nuclei from them. You can remove large clumps by filtration or density gradient centrifugation. [7]
You can also modulate the osmotic pressure of the extraction buffer by adjusting the concentration of osmolytes such as sucrose.
If your nuclei can tolerate it, balance the electrostatic interactions between nuclei by adding/adjusting the concentration of salts such as NaCl and MgCl2.
This post on 10x Genomics further highlights some best practices for nuclei isolation.
Q: What is the accuracy range of the CellDrop?
A: The CellDrop™ offers a flexible sample range from as few as 700 – 25,000,000 cells per mL, depending on the sample arm height.
At the default sample arm height of 100 µm, the accuracy range is 100,000 – 10,000,000 cells per mL.
The lower arm height (50 µm) allows the use of lower measurement volumes to conserve precious sample.
Q: I need to inspect my nuclei under a high-resolution microscope and count them. Can I use the microscope slides on the CellDrop™ to reduce sample wastage?
A: Yes. While the CellDrop™ does not require microscope slides to count nuclei, the sample arm can be set high enough to accommodate them, so you can use them on the CellDrop™ to cut down on the sample volume needed for nuclei counting and analysis.
References
- Jovic D, Liang X, Zeng H, Lin L, Xu F, Luo Y. (2022). Single-cell RNA sequencing technologies and applications: A brief overview. Clin Transl Med. 12(3):e694
- Habib N, Avraham-Davidi I, Basu A, et al. (2017). Massively parallel single-nucleus RNA-seq with DroNc-seq. Nat Methods 14:955–58
- Buenrostro J, Wu B, Litzenburger U, et al. (2015). Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523:486–90
- Strober W. (2015). Trypan Blue Exclusion Test of Cell Viability. Curr Protoc Immunol 111:A3.B.1-A3.B.3
- AAT Bioquest, Inc. (2024, April 12). Quest Graph™ Spectrum [Acridine Orange]. AAT Bioquest. Available at: https://www.aatbio.com/fluorescence-excitation-emission-spectrum-graph-viewer/acridine_orange. Accessed April 12, 2024
- AAT Bioquest, Inc. (2024, April 12). Quest Graph™ Spectrum [Propidium iodide]. AAT Bioquest. Available at: https://www.aatbio.com/fluorescence-excitation-emission-spectrum-graph-viewer/propidium_iodide. Accessed April 12, 2024
- Katholnig K, Poglitsch M, Hengstschläger M, Weichhart T. (2015). Lysis gradient centrifugation: a flexible method for the isolation of nuclei from primary cells. Methods Mol Biol. 1228:15–23
