Nuclei isolation for single-nuclei rna sequencing for larger tissues

Technical
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1

Hi,

From your protocl - Isolation of Nuclei from Adult Human Brain Tissue for 10x Genomics Platform

It mentions that if the tissue is larger than 100mg, 7ml grinder is used. What is the upper limit for it? I have tissue blocks weight between 0.8g to 1g. Will 7ml grinder be sufficient, if not, can you recommend me a size, please?

And apart from the volume of homogenization buffer used differing between the small tissue and large tissue, does everything else stay the same? And if I need a bigger douncer (when 7ml grinder is not sufficient), will it be applicable for me to follow the protocol as it is and just change the homogenisation buffer at the beginning?

Thank you!

Best,
Kawai

2

Hi Kawai,

Your tissue blocks should work fine in a 7ml Dounce homogenizer. Since you are using larger pieces of tissue than we outline in the protocol, my recommendation would be to increase the volumes of all of the solutions accordingly. So, in addition to increasing the homogenization buffer volume, I would recommend increasing the volume of blocking buffer. If you are going to proceed to run flow cytometry you will want to take care not to clog your instrument and dilute your sample accordingly. Let us know if you have any follow up questions.
Best,
Rebecca