Question: How can I assess the quality of my nuclei for Single Cell ATAC Sequencing?
Answer: A high-quality nuclei suspension is critical for Single Cell ATAC sequencing. There are a couple of ways to asses 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 Protocol 'Nuclei Isolation for Single Cell ATAC Sequencing.' 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).
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?"
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