Question: How can I improve my nuclei recovery using the Chromium Nuclei Isolation kit?
Answer: Nuclei yield for the Chromium Nuclei Isolation kit can vary depending on tissue type, cell content, and disease state. Nuclei yields for healthy tissues typically range from 5-15k nuclei/mg.
Factors impacting nuclei yield:
- Starting tissue mass: The nuclei yield increases proportionally with input tissue mass for most tissues. For tissue mass inputs outside the supported range (<3mg), nuclei yield may be reduced due to less efficient nuclei pelleting, and more loss during washes and spins.
Tissue type: Sample quality and cell density for tissue types will determine nuclei yield.
Recommendations for improving nuclei yields or when working with limited samples:
- Use a swing bucket rotor: Nuclei will pellet at the bottom of the tube in a tighter pellet, reducing loss during washes
- Perform a reduced number of washes: The protocol includes two washes with Wash and Resuspension Buffer; for low yield samples, performing a single wash may improve yield. Note: There is a trade-off between debris and nuclei yield; evaluate how much debris and yield you observe after a single wash vs. two washes to determine what is best for your sample.
- Leave some buffer behind: The User Guide recommends leaving up to 200uls behind during washes with Wash and Resuspension Buffer; this helps retain nuclei so that they are not washed away.
Increase centrifugation times: Centrifugation time may be increased from 5 to 10 minutes during wash steps only. This may improve nuclei pelleting and reduce sample loss during wash steps.
Products: Single Cell Gene Expression, Single Cell Immune Profiling, Single Cell ATAC, Single Cell Multiome ATAC + GEX, Fixed RNA Profiling