Cell isolation from dorsal mouse skin for single-cell RNA-seq

Dispase Solution

Dissolve 0.5g ofin a 50ml ofto make 10mg/mL of Dispase II solution

Filter the solution by using a 0.2µm filter

Aliquot 1.25mL in tubes and store at -20°C

Collagenase type I Solution

Dissolve 1g ofin 4mL of to make a 250mg/mL collagenase type I solution

Make a stock solution

i.e. 67500U/mL x 4mL = 50U/µL x V_max

V_max= 5.4mL

5.4 - 4.0 = 1.4mL to add the stock to make the stock solution

Filter the solution by using a 0.2µm filter

Aliquot 20µL in tubes and store at -20°C

Liberase TM Solution

Punch through the cap with a syringe to add 2mL of into 5mg bottle to make 2.5mg/mL liberase solution

Dilute into 0.2mg/mL in (1.04 Wünsch unit/mL)

Filter the solution by using a 0.2µm filter

Aliquot 750µL in tubes and store at -20°C

Working Solution 1 (WS1)

Make a 0.5Mass / % volume ofin 50mL of

Filter the solution by using a 0.2µm filter and keep it at 4°C

Working Solution 2 (WS2)

Make a 0.04Mass / % volume of Bovine serum albumin solution in 50mL of

Filter the solution by using a 0.2µm filter and keep it at 4°C

Enzyme Cocktail 1 (EC1)–4 samples

From the enzyme stocks that have been prepared before, mix:

• 1.25mL of Dispase
• 0.75mL of Liberase
• 20µL of type I Collagenase in a 15mL tube

Add 8.0 mL of

Warm up in a 37°C water bath before using

Enzyme Cocktail 2 (EC2)–4 samples

Dilute the in to make a 0.05Mass / % volume of trypsin/EDTA solution

Warm up in a 37°C water bath before using

Punch out the skins from mice using a sterileand immediately float on WS1 on ice

Peel off any fat tissue beneath the skin using a blade

Mince the fat tissue-cleaned skin samples into smaller pieces using sterile scissors while holding the tissue with sterile forceps on ice

Incubate the minced tissues in 2 ml of EC1 in a 6-well plate at a 37°C incubator with a 5% CO₂ supplementation for two hours

2h 0m 0s

Shake the plate orbitally in the incubator every 0h 15m 0s for better digestion

Remove a piston from a sterile,to smash the partly-digested tissues in the plate

Place ainto a 50mL tube and sift through the cell/tissue mixture

Wash the EC1-digested tissue on the cell strainer with WS1 thrice

Save the flow-through on ice

Invert the cell strainer with tissue remnants and place it on a sterile 6-well plate

From the sieve-through side of the cell strainer, add 2mL of EC2 aiming at tissue remnants

Incubate the undigested tissue at a 37°C incubator with a 5% CO₂ supplementation for 0h 15m 0s

Remove a piston from a sterile,to smash the partly-digested tissues in the plate

Place a sterileinto the 50mL tube obtained at Step 15 to pool and sift through the cell/tissue mixture

Wash the trypsin-digested tissue on the cell strainer with WS1 thrice

Place a sterileinto a clean 50mL tube and sift through the cell suspension

Centrifuge the pooled cell suspensions at 300x g,20°C

Carefully aspirate the supernatant and resuspend the pellet in 1mL of WS2

Centrifuge the pooled cell suspensions again at 300x g,20°C

Carefully aspirate the supernatant and resuspend the pellet in 500µL of WS2

Determine the cell viability by mixing 10µl of the cell suspensions with 10µl of trypan blue

Equipment

Value	Label
Countess 3 FL Automated Cell Counter	NAME
Automated Cell Counter	TYPE
Thermofisher scientific	BRAND
AMQAF2000	SKU

Take at least four counts per sample

Remember to tick "Trypan Blue correction" on the Countess cell counter

Average cell numbers for each sample and dilute each cell suspension with WS2 to adjust the cell number to 700-1200 cells/µL as indicated in 10X Genomics Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 Protocol (CG000315)

Aim for cell viability above 70% for each sample

If the cell viability is below 70%, you may choose to remove dead cells using a dead cell removal kit

Immediately proceed with Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 Protocol (CG000315)

Dispase Working Solution