General Manual Tissue Dissociation Protocol

When performing this manual tissue dissociation protocol, be sure to handle liquid nitrogen safely and use appropriate protection against the extremely cold metal.

1. Prepare EpiCypher Nuclei Extraction Buffer (NE Buffer) by adding spermidine and protease inhibitor as per the recipe below:

   For 10mL of Complete EpiCypher Nuclei Extraction Buffer:

   1. Resuspend one large EDTA-free complete protease inhibitor tablet (EpiCypher 21-1027-L) in 2mL of molecular biology grade water to create a 25X stock.

   2. Add 9.6mL of pre-nuclei extraction buffer (from EpiCypher 21-1026) to a conical tube.

   3. Add 0.4mL of 25X protease inhibitor (made in step a) above) to the tube.

   4. Add 5uL 1M spermidine (from EpiCypher 21-1026) (final concentration 0.5mM).

   5. Keep on ice.
      

2. Prepare the biopulverizer and spatulas by cooling down all parts in liquid nitrogen for 5 minutes, then remove and place on a clean bench space. Small biopulverizers will remain cold for a few minutes, while larger plates will remain cold for up to 30 minutes. If processing multiple samples, be sure to clean and re-cool your biopulverizers so as not to thaw any of your samples.

3. Place the frozen tissue piece in the well of the biopulverizer. Place the pestle on top and hit the pestle with the mallet several times while holding the pestle on top of the tissue piece. Your tissue should be fragmented into tiny pieces resembling sand.

4. With your spatula, scrape the pulverized tissue into a tube and store on dry ice or at -80C until you have processed all samples.

5. Add 1-2 mL of NE Buffer per <=100 mg of pulverized tissue and incubate on ice for 10 minutes.

6. Pipette up and down several times before filtering your sample through a 70 um filter into a 15 mL conical tube.

7. Centrifuge the 15 mL conical tube at 600 x g for 5 minutes at 4 ˚C.

8. You should see a clear nuclei pellet in your tube. Remove the supernatant and resuspend in 1-2 mL of NE Buffer or PBS.

9. Stain a small sample of your nuclei for quality assessment & quantification. For tissue, we recommend propidium iodide—see more in this article.

10. Cryopreserve nuclei in nuclei extraction buffer (see steps here) or proceed directly to your CUTANA assay.