Question: How can I assess my nuclei's quality for Single Cell ATAC or Single Cell Multiome ATAC+GEX Sequencing?
Answer: A high-quality nuclei suspension is critical for Single Cell ATAC sequencing or Single Cell Multiome ATAC+GEX Sequencing. There are a couple of ways to assess the quality of a nuclei preparation:
- Cell Viability Measurements: Trypan blue, along with an automated counter or a hemocytometer, can be used 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. Example cell viability measurements and images are available in the Demonstrated Protocols 'Nuclei Isolation for Single Cell ATAC Sequencing' and 'Nuclei Isolation from Mouse Brain Tissue for Single Cell ATAC Sequencing'. The Cellaca MX FL5 and Luna-FL are instruments that support counting isolated nuclei. Isolated nuclei have also been counted using Countess II FL and NucleoCounter NC-202 with success. Manual counting using hemocytometer is also recommended. Countess 3 FL is currently incompatible with isolated nuclei counting due to an updated counting algorithm which is more aggressive at gating debris, defined as particles <4uM.
Note: For samples with debris, ethidium homodimer-1 or other fluorescent dyes may help distinguish nuclei from debris for accurate quantitation.
- Visualization with Microscopy: The nuclei may also be assessed by visualization under a microscope. A good nuclei suspension will show clump-free, debris-free nuclei. Using a high-powered microscope (at least 40x), it may be possible to visualize individual nuclei to assess the membrane quality. A nucleus with an intact membrane should appear round and smooth (Panel A). A nucleus with a compromised membrane will appear “ruffled” and show evidence of blebbing (Panels B-D). Blebbing is the loss of the nuclear membrane's coherence with the nucleoplasm and is normally caused by over lysis. In general, users need to optimize the lysis protocol.
If using the Chromium Nuclei Isolation Kit to isolate nuclei, no higher magnification QC to assess nuclear membrane integrity is required for pilot experiments or working with a new tissue type. If using the Chromium Nuclei Isolation Kit, the following article reviews nuclei counting and QC: How should I count nuclei after isolating nuclei using the Chromium Nuclei Isolation Kit?
To learn how to optimize nuclear isolation for Single Cell ATAC, refer to the article "How can I optimize my nuclei prep for Single Cell ATAC sequencing."
To learn how to optimize nuclear isolation for Single Cell Multiome ATAC+GEX, refer to the article "What are the best practices for nuclei isolation for Single Cell Multiome ATAC and Gene Expression?"
A: High-quality nuclei have well-resolved edges. Optimal quality for single-cell ATAC libraries.
B: Mostly intact nuclei with minor evidence of blebbing. Quality single-cell ATAC 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!
Product: Single Cell ATAC, Single Cell Multiome ATAC + GEX