After euthanizing mouse, remove hair using Nair: dab with Nair, wait 30 secs, wipe with wet paper towel.

Isolate tissue and place in ice-cold hypothermosol.

Scrape off underlying layer of fatty / connective tissue using scalpel before proceeding.

Mince skin tissue thoroughly on petri dish on ice for 3-4 min on ice into 1-mm3 pieces using razor blade while manipulating tissue with forceps – you will need to use grinding motion and vigorously break up tissue.

0h 4m 0s

Place 23 mg minced tissue into 1 mL B. Lich enzyme cocktail. Incubate on ice.

23mg

Shake every min; triturate 10x every 2 min with p1000 w/tip cut (start triturating at 2 min) for 20 min.

0h 20m 0s

0h 1m 0s``0h 2m 0s

After 20 mins of triturating on ice, use pipet to transfer digest mix to 2 mL dounce homogenizer. Use 10 strokes of Pestle A every 2 min (4 series total, 8 min). Digest mix should become turbid.

0h 2m 0s``0h 8m 0s

Transfer back to 1.5 mL tube using 1 mL serological pipet. Mix thoroughly and allow to settle on ice 2 min.

0h 2m 0s

Save 70% (700 µL) of supernatant, leaving chunks at the bottom of the tube; apply to 30 µM filter on 15 mL conical. Rinse filter w/5 mL ice-cold PBS/BSA 0.04%. Save flow through on ice and keep filter on tube for 2nd layer.

700µL``5mL

Add additional 1 mL b. Lich enzyme mix to residual tissue chunks.

1mL

Triturate 10x every 2 min, shake every min while incubating on ice for 20 additional mins. (50 min. total digest time).

0h 20m 0s

0h 2m 0s``0h 1m 0s

Transfer entire volume to same 30 µM filter on 15 mL conical. Rinse with additional 5 mL ice-cold PBS/BA 0.04%.

5mL

Centrifuge at 300 g for 5 min at 4 °C. Remove supernatant & re-suspend in 100 µL PBS/BSA 0.04%. Examine using hemocytometer with trypan blue.

0h 5m 0s``100µL

Adjust concentration to 1,000 cells/µL for Chromium or 100 cells/µL for DropSeq.