Question: What are the best practices for nuclei isolation for Single Cell Multiome ATAC and Gene Expression?
Answer: Here are some of the best practices while working with nuclei samples for the Single Cell Multiome ATAC and Gene Expression assay:
It is recommended that lysis times of nuclei are optimized so as not to overlyse the nuclear membrane. Overlysed nuclei tend to stick together and form clumps. Moreover, overlysis may lead to the leakage of nuclear content leading to a high level of background in your data.
In order to optimize lysis, it's best to perform a lysis timeline as described in this article: How do I perform a lysis timeline to optimize my nuclei isolation for Single Cell ATAC sequencing? (although this article mentions Single Cell ATAC, the same guidelines for running a lysis timeline for Single Cell Multiome ATAC and Gene Expression apply)
To assess lysis efficiency, use Trypan blue along with an automated counter or a hemocytometer to measure the sample's viability. Unlysed cells will stain as live while nuclei will stain as dead. Measurements of <5% live input cells indicate proper cell lysis. Improper lysis is denoted by an abundance of live cells, meaning cell membranes have not been lysed and nuclei have not been isolated. Note: For samples with debris, ethidium homodimer-1 or other fluorescent dyes may help distinguish nuclei from debris for accurate quantitation.
After nuclei isolation, the nuclear membrane should also be visualized under a microscope at 40x or 60x magnification to ensure no blebbing. Below are some great visuals on how to quality check the nuclear membrane :
A: High-quality nuclei have well-resolved edges. Optimal quality for single-cell gene expression libraries.
B: Mostly intact nuclei with minor evidence of blebbing. Quality single-cell gene expression libraries can still be produced.
C: Nuclei with strong evidence of blebbing. Proceed at your own risk.
D: Nuclei are no longer intact. Do not proceed!
Debris and Clumping :
Nuclei clumping could be a sign of overlysis. This can be avoided by using optimal lysis conditions(see above). Nuclei suspensions can also be filtered to reduce clumps. See: What cell strainers are recommended?
Sorting of nuclei to remove debris can be performed, however, extra care should be taken to avoid nuclei damage as additional handling of nuclei can be stressful and further compromise the nuclear membranes, leading to leakage of nuclear content. Additionally, certain sorting dyes can alter chromatin structure: Can I sort nuclei for Single Cell ATAC sequencing? Follow recommended sorting guidelines as described in the nuclei isolation demonstrated protocols.
Low Nuclei Recovery:
In cases where low nuclei recovery is an issue, centrifugation time may be increased in an attempt to improve recovery. The use of swing-bucket rotors has also improved final nuclei recovery in some instances.
Nuclei being smaller in size may be more difficult to count. This can be further exacerbated if there is lots of cellular debris present, as common counting dyes like Trypan Blue may also stain debris. The use of a nucleic acid staining fluorescent dye (like Ethidium Homodimer-1) and a fluorescent capable automated counter or microscope may help improve nuclei counting accuracy as only nucleic acids will be stained and not debris. It is best to count in replicates to ensure accuracy. Please see our Technical Note with Guidelines on Accurate Target Cell Count
Freezing or cryopreservation of nuclei is not recommended as freezing can damage cellular/nuclear membranes. Furthermore, since nuclei are permeabilized, freezing can increase the potential of nuclear leakage and increase the background signal. Cryopreservation at the cell or tissue stage is recommended as an alternative to the freezing of nuclei.
For protocols to isolate nuclei for Single Cell Multiome ATAC and Gene Expression, refer to the following demonstrated protocols: https://support.10xgenomics.com/single-cell-multiome-atac-gex/sample-prep
Products: Single Cell Multiome ATAC and Gene Expression