What are the best practices for working with nuclei samples for 3' single-cell gene expression?

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:

 

Lysis Conditions:

When isolating nuclei using the Chromium Nuclei Isolation Kit, lysis optimization is not recommended with new sample types: Do I need to optimize any lysis conditions when using the Chromium Nuclei Isolation Kit?

Alternatively, if using a demonstrated protocol 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 can I run a lysis timeline to optimize nuclei isolation for 3' single-cell gene expression?

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.

Buffer: 

While working with nuclei samples, it is critical to have RNAse inhibitors in the lysis, wash and resuspension buffer. For the 3'GEX assay, we recommend a concentration of 0.2U/ul of RNAse inhibitor.

Nuclei Purification: 

There are two methods to purify nuclei after isolation:

• FACS sorting:  helps remove subcellular debris, aggregates/clumps and ambient RNA/DNA. Forward and side scatter should be used to exclude nuclei doublets, triplets and other clumps. For an unbiased nuclei population, sorting may be completed using the DAPI+ population. The sorted single-nuclei solution should be used immediately as input to the 10x 3’ Single-Cell Gene Expression assay. Extra care should be taken to avoid nuclei damage as sorting can be stressful and compromise the nuclear membranes, leading to leakage of nuclear content. See : What are the best practices for flow sorting cells for 10x Genomics assays?
• Density gradient using Iodixanol (OptiPrep™) or a modified sucrose gradient: Both of these methods can be used for cleaning up a nuclei prep, however, as reported in literature, iodixanol gradients are preferred over sucrose gradients. Sucrose solutions are more viscous and hyperosmotic, while iodixanol is isosmotic, allowing nuclei isolation under iso-osmotic conditions. Nuclei should be washed 3x and resuspended in an appropriate buffer after using an Optiprep gradient to ensure all iodixanol has been removed.

With both these methods, about 50% or more of nuclei loss is expected, so there should be a sufficient number of nuclei to start with.

 

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.

 

Counting:

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.

To enable nuclei counting on Countess 3 FL, enable “FL-based count.” BF-based counting may gate out cells/nuclei <4um. For more information on how to accurately count nuclei, please see: I'm having trouble accurately counting nuclei. What can I do?

 

Storage:

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.

 

Workflow:

For 3' v3.1, 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 3' v4, no protocol adjustments are recommended. If running nuclei for the first time, you may consider running a positive control, such as a cultured cell line with high viability (>90%) and yielding a sufficient number of nuclei (i.e. >1 million nuclei). 

 

Software: 

For analyzing 3' Gene Expression data from nuclei samples, please follow the following guidelines:

• If you use version 5 or later of Cell Ranger, please run cellranger count  with --include-introns to count reads mapping to intronic regions.
• If you use an older version of Cell Ranger, then you need to create a 'pre-mRNA' transcriptome reference for cellranger count in order 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