Question: What are the best practices for working with nuclei samples for 3' single-cell gene expression?
Answer: Here are some of the best practices while working with nuclei samples for the 3' Gene Expression Assay:
It is recommended that lysis times of nuclei are optimized so as not to overlyse the cells. Overlysed cells(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 apply to our 3' Single Cell Gene Expression assay as well)
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 sticking together 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?
The concentration of BSA in the wash and resuspension buffer can be increased up to 2% to reduce clumping.
Sorting of nuclei to remove clumps 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.
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. If nuclei are frozen, they have the potential to burst and increase the background signal. Nuclei can be frozen, but lots of sample loss is expected (due to membranes bursting). For details, see: Is it possible to freeze nuclei prior to Single Cell 3'? Cryopreservation at the cell stage is recommended as an alternative to the freezing of nuclei.
As nuclei have lower RNA content than whole cells, increase the cDNA amplification cycles(Step 2.2 in the user guide) by 1-2 cycles to increase the cDNA yield. No other changes are required in the 10x protocol while using nuclei instead of cells.
For analyzing 3' Gene Expression data from nuclei samples, it maybe useful to use a "pre-mRNA" transcriptome reference for cellranger count if you would like to include reads mapping to intronic regions in the UMI count. For more information on creating a pre-mRNA reference package, please see the support site: Making pre-MRNA references
For protocols to isolate nuclei for 3' single-cell gene expression, see: How can I isolate nuclei for 3' gene expression profiling?
Products: Single Cell Gene Expression