Nuclear Extraction from Tissue for FACS

Coat all tubes (centrifugation, sample and collection tubes) in coating medium (1% (v/v), filtered).

Cool ultracentrifuge to 4°C prior to centrifugation

Pre-weigh the DTT needed.

Keep douncers On ice.

Put 2.8mL in centrifuge tube.

Mix 6mL + 2mL in 15 ml falcon tube.

If the tissue is large (>2 mm3), chop the tissue with a razor.

Collect chopped tissue with 0.5mL and transfer to douncer. Repeat with another 0.5mL to get all tissue.

Dounce: 5-7x loose, 5-7x tight.

Pour douncer content into falcon tube with sucrose/lysis buffer mixture and flip upside down a few times. Pour 1mL of tube content back and forth from douncer to collect remaining nuclei.

Carefully layer the 8.5mL to the centrifuge tube, on top of sucrose.

Balance the tubes (≤5mg difference).

Place tubes in ultracentrifuge, spin at 15500rpm,0h 0m 0s.

Carefully aspirate off supernatant without disturbing the pellet.

Soften pellet for 0h 10m 0s in 50µL.

Add 100µL (containing a nuclei marker, such as Draq7) and filter using 70um pipette tip filters into coated tubes.

FACS sort, low flowrate, largest nozzle, cooling system on. The sucrose will cause problems at FACS. Avoid this by adding dilution buffer (with your nuclei marker), 100µL at the time (usually need 300µL-400µL for dilution to avoid problems – add that even before starting to sort).

For single nuclei sequence using 10x: Pre-coat the pipette tips needed to load the 10x machine according to step 1.

For RNA extraction: Pellet nuclei at 1300x g,0h 0m 0s. Remove supernatant and snap freeze for later RNA extraction (or resuspend in 350µL directly. Use RNeasy micro columns. For very small volumes, resuspend in RLT without pelleting).