Nuclei isolation for single-nuclei rna sequencing

I am a research technician working in the field of neurology and I am currently trying to isolate nuclei from frozen human brain samples (prefrontal cortex). I would like to use these nuclei for single-nuclei RNA seq.
The procedure i am following can be summarized as follows: i homogenize the tissue (around 25 mg) in a dounce homogenizer and then do a clean-up with ultracentrifugation. I was wondering if someone is using a similar approach (but even other approaches for the clean-up phase such as FACS) and could share with me how many nuclei (total number) are obtained before and after the clean-up step.

I was alao wondering how many nuclei are loeaded on the chromium controller for GEM generation. The 10x guidelines for single cell sequencing suggest to load a maximum of ca 16500 cells in order to target 10000 cells. Do these numbers also apply to nuclei or is it necessary to load a higher number of nuclei?

thank you for your help .)

Martina

Hi Martina,

Thanks for your question. We use the following protocol for nuclei isolation from human brain:

This protocol involves dounce homogenization followed by optional cleanup of the sample with myelin removal beads. We also perform FACS using NeuN antibody to select for neurons and non-neuronal nuclei. We don’t explicitly quantify the nuclei before and after myelin removal cleanup. For 10x loading, we follow the guidelines provided by 10x and typically load our ports at max (~16500 nuclei). We typically recover ~9000 nuclei per 10x port. Let us know if you have any other questions.

-Rebecca