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 .)