TITLE: Cell isolation from dorsal mouse skin for single-cell RNA-seq
AUTHORS: Cenk Celik, Stella Yue Ting Lee, Guillaume Thibault

[DESCRIPTION]
We identified heterogeneity of the skin cell populations upon Enterococcus faecalis infection.

[STEPS]
SECTION: Enzyme solutions
50. Dispase Working Solution
SECTION: Enzyme solutions
0.1. Dispase Solution

Dissolve 0.5g of in a 50ml of to make 10 mg/mL of Dispase II solution

SECTION: Enzyme solutions
0.2. Filter the solution by using a 0.2µm filter
SECTION: Enzyme solutions
0.3. Aliquot 1.25mL in tubes and store at -20 °C
SECTION: Enzyme solutions
1. Collagenase type I Solution

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

SECTION: Enzyme solutions
1.1. Make a stock solution

i.e. 67500U/mL x 4mL = 50U/µL x Vmax
Vmax = 5.4mL
5.4 - 4.0 = 1.4mL to add the stock to make the stock solution
SECTION: Enzyme solutions
1.2. Filter the solution by using a 0.2µm filter
SECTION: Enzyme solutions
1.3. Aliquot 20µL in tubes and store at -20 °C 

SECTION: Enzyme solutions
2. Liberase TM Solution

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

SECTION: Enzyme solutions
2.1. Dilute into 0.2 mg/mL in    (1.04 Wünsch unit/mL)

SECTION: Enzyme solutions
2.2. Filter the solution by using a 0.2µm filter
SECTION: Enzyme solutions
2.3. Aliquot 750µL in tubes and store at -20 °C 

SECTION: Working Solutions
3. Working Solution 1 (WS1)

Make a 0.5 Mass / % volume of in 50mL of   
SECTION: Working Solutions
3.1. Filter the solution by using a 0.2µm filter and keep it at 4 °C 

SECTION: Working Solutions
4. Working Solution 2 (WS2)

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

SECTION: Working Solutions
4.1. Filter the solution by using a 0.2µm filter and keep it at 4 °C 
SECTION: Working Solutions
5. 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
SECTION: Working Solutions
5.1. Add 8.0 mL of
  

SECTION: Working Solutions
6. Enzyme Cocktail 2 (EC2)–4 samples

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

SECTION: Working Solutions
5.2. Warm up in a 37 °C water bath before using
SECTION: Working Solutions
6.1. Warm up in a 37 °C water bath before using
SECTION: Tissue Dissociation and Cell Isolation Procedure
7. Punch out the skins from mice using a sterile and immediately float on WS1 on ice

SECTION: Tissue Dissociation and Cell Isolation Procedure
8. Peel off any fat tissue beneath the skin using a blade  
SECTION: Tissue Dissociation and Cell Isolation Procedure
9. Mince the fat tissue-cleaned skin samples into smaller pieces using sterile scissors while holding the tissue with sterile forceps on ice
SECTION: Tissue Dissociation and Cell Isolation Procedure
10. 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

120 min 
SECTION: Tissue Dissociation and Cell Isolation Procedure
11. Remove a piston from a sterile, to smash the partly-digested tissues in the plate
SECTION: Tissue Dissociation and Cell Isolation Procedure
12. Place a into a 50mL tube and sift through the cell/tissue mixture

SECTION: Tissue Dissociation and Cell Isolation Procedure
13. Wash the EC1-digested tissue on the cell strainer with WS1 thrice
SECTION: Tissue Dissociation and Cell Isolation Procedure
14. Save the flow-through on ice
SECTION: Tissue Dissociation and Cell Isolation Procedure
15. Invert the cell strainer with tissue remnants and place it on a sterile 6-well plate
SECTION: Tissue Dissociation and Cell Isolation Procedure
16. From the sieve-through side of the cell strainer, add 2mL of EC2 aiming at tissue remnants
SECTION: Tissue Dissociation and Cell Isolation Procedure
17. Incubate the undigested tissue at a 37 °C incubator with a 5% CO2 supplementation for 15 min
SECTION: Tissue Dissociation and Cell Isolation Procedure
18. Remove a piston from a sterile, to smash the partly-digested tissues in the plate
SECTION: Tissue Dissociation and Cell Isolation Procedure
19. Place a sterile into the 50mL tube obtained at Step 15 to pool and sift through the cell/tissue mixture
SECTION: Tissue Dissociation and Cell Isolation Procedure
20. Wash the trypsin-digested tissue on the cell strainer with WS1 thrice
SECTION: Tissue Dissociation and Cell Isolation Procedure
21. Place a sterile into a clean 50mL tube and sift through the cell suspension

SECTION: Tissue Dissociation and Cell Isolation Procedure
22. Centrifuge the pooled cell suspensions at 300 x g, 10 min, 20 °C 
SECTION: Tissue Dissociation and Cell Isolation Procedure
23. Carefully aspirate the supernatant and resuspend the pellet in 1mL of WS2
SECTION: Tissue Dissociation and Cell Isolation Procedure
24. Centrifuge the pooled cell suspensions again at 300 x g, 5 min, 20 °C
SECTION: Tissue Dissociation and Cell Isolation Procedure
25. Carefully aspirate the supernatant and resuspend the pellet in 500µL of WS2
SECTION: Tissue Dissociation and Cell Isolation Procedure
26. Determine the cell viability by mixing 10µl of the cell suspensions with 10µl of trypan blue 
 

SECTION: Tissue Dissociation and Cell Isolation Procedure
26.1. Take at least four counts per sample
SECTION: Tissue Dissociation and Cell Isolation Procedure
26.2. Remember to tick "Trypan Blue correction" on the Countess cell counter
SECTION: Tissue Dissociation and Cell Isolation Procedure
27. 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)
SECTION: Tissue Dissociation and Cell Isolation Procedure
28. Immediately proceed with Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 Protocol (CG000315)
SECTION: Tissue Dissociation and Cell Isolation Procedure
27.1. Aim for cell viability above 70% for each sample
 

SECTION: Tissue Dissociation and Cell Isolation Procedure
27.2. If the cell viability is below 70%, you may choose to remove dead cells using a dead cell removal kit
SECTION: Tissue Dissociation and Cell Isolation Procedure
10.1. Shake the plate orbitally in the incubator every 15 min for better digestion