General Automated Tissue Dissociation Protocol

IMPORTANT NOTE: This protocol is for frozen tissue. For fresh tissue, we recommend either 1) freezing it down and then continuing with this protocol, or 2) using the GentleMACS using one of the preset programs for your specific tissue with appropriate enzyme cocktail. Once you have obtained a single-cell suspension, you can move into nuclei extraction for your specific assay.

Where possible, it is recommended to use this automated protocol as opposed to the manual tissue dissociation protocol, as the automation reduces handling and allows for uniform dissociation.

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

   For 10 mL of Complete EpiCypher Nuclei Extraction Buffer:

   1. Resuspend 1x large EDTA-free complete protease inhibitor tablet in 2mL of molecular biology grade water to create a 25X stock.

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

   3. Add 0.4 mL of 1X protease inhibitor (created in step a) above) to the tube.

   4. Add 5 uL 1M spermidine (final concentration 0.5mM).

   5. Keep on ice.

2. Add 1-2 mL of NE Buffer per 100 mg or less of frozen tissue to a Miltenyi GentleMACSTM C-tube.

3. Add the frozen tissue biopsy and immediately load C-tube upside down on the GentleMACS Tissue Dissociator ensuring that the tissue is submerged in the liquid.

4. Add Octo Coolers and run the preset 4C_Nuclei_1 Program.

5. Remove C-tube from the machine.

   1. If bubbles have formed, it is possible to collect the liquid at the bottom of the tube with a quick centrifugation at 300 x g for 1 minute.

   2. If your tissue is not fully dissociated, re-run the program.

6. Filter your sample through a 70 um filter into a 15 mL conical tube and wash out the C-tube with 5 mL of PBS.

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. Remove the supernatant and resuspend in 1-2 mL of Nuclei Extraction Buffer or PBS.

9. Stain a small sample of your nuclei for quality assessment & quantification. Our recommendation for staining is to use propidium iodide.

10. Cryo-preserve nuclei in Nuclei Extraction Buffer or proceed to your CUTANA assay.