TITLE: Human endometrium and endometriosis tissue dissociation for single-cell RNA sequencing
AUTHORS: Yuliana Tan, Diane Luo, Suleyman Bozal, Paul Robson, Elise Courtois

[DESCRIPTION]
Tissue dissociation is critical step to ensure the quality of samples for single-cell RNA sequencing. To ensure recovery of most cells in the tissue without introducing stress response from the dissociation process, we adapted the dissociation method by Adam et al. (2017) and optimized for the dissociation of human endometrium and endometriosis tissue (both ectopic peritoneal and ovary lesions), targeting high quality viable cells (above 70%) with high transcript and gene counts per cell.

[BEFORE_START]
Coat all tubes and tips with serum buffer, e.g. 2.5% BSA or "Buffer I" used in this protocol
Ensure centrifuge is set to 4°C.
Prepare cold water bath (6°C). 
 

 



[GUIDELINES]
This protocol was adapted from Adam et al. (2017) and optimized for dissociation of human endometrium and endometriosis tissue (from ectopic peritoneal and ovary lesion) for single-cell RNA sequencing, yielding high UMI and gene counts per cell.  

Tissue was collected fresh from the operating room and immediately submerged in MACS Storage Buffer before transport to laboratory on ice. Processing of tissue shortly after receiving will ensure high cell viability and (hopefully) high quality single-cell RNA sequencing data.
 


[STEPS]
SECTION: Buffer and Enzymes Preparation
1. 
Prepare fresh digestion enzyme - Cold Active Protease (CAP) solution:
 

SECTION: Viable Cell Sorting
21. Stain cells with viability dye (Propidium Iodide and Calcein Violet) following manufacturer's protocol, incubate 20 min.

 
 

SECTION: Tissue Digestion with CAP
7. Mince the tissue to 1 mm2 cube with sterile scalpel. 
SECTION: Tissue Digestion with CAP
8. Add 2 mL into the dish.

SECTION: Tissue Digestion with CAP
9. Transfer all materials from the dish to GentleMACS C Tubes using wide-bore tips, then incubate in cold water bath for 7-10 minutes. 
(Coat the   with 2.5 % (v/v) before use)

SECTION: Tissue Digestion with CAP
10. Place tube on gentleMACS Dissociator and run “mouse_brain_3” protocol. Repeat twice (2x).
 

SECTION: Tissue Digestion with CAP
11. When the run is finished, place the tube in cold water bath and let tissue settle down for 30 s1 min 


SECTION: Tissue Digestion with CAP
12. Transfer 1-1.5 mL supernatant containing dissociated single-cells to 50 mL conical tube containing 30 mL.
Do not disturb the non-dissociated tissue fragments at the base of the tube. 

CRITICAL STEP: Keep collected cell suspension on ice until the tissue dissociation procedure is completed. 
SECTION: Tissue Digestion with CAP
9.1. Triturate the mixture with 1000 µL wide-bore tips for 15 s every 2 min to ensure homogenization and dissociation.
 

SECTION: Tissue Digestion with CAP
13. Add another freshly made 2 mL  into gentleMACS C Tubes containing the remaining non-dissociated tissue fragments. Incubate in cold water bath for  20 min 40 min.



SECTION: Tissue Digestion with CAP
13.1. Triturate the mixture with 1000 µL wide-bore tips for 15 s every 5 min to ensure homogenization and dissociation.
SECTION: Tissue Digestion with CAP
13.2. Place tube on gentleMACS Dissociator and run “mouse_brain_3” protocol every 15 min
 


SECTION: Setup for Cell Capture on 10x Chromium
24. Load 12,000 cells using the Chromium Next GEM Single Cell 3-prime kit v3.1, onto a 10x Chip G and process according to manufacturer's protocol (CG000315 Rev A).
  (cat#) onto 10x Chip (cat#), rocess according to manufacturer's protocol.
  
   cells using the Chromium Next GEM Single Cell 3-prime kit v3.1, onto a 10x Chip G and process according to manufacturer's protocol (CG000315 Rev A.


SECTION: Tissue Digestion with CAP
14. Repeat  . Transfer the supernatant to the 50 ml conical tube containing single cells from step 8.
SECTION: Tissue Digestion with CAP
15. Observe the leftover tissue fragments under microscope. Leftover tissue fragments may contain fibers (extracellular matrix) and/or cells.

If there are few or no cells attached,  .
otherwise, follow the step below. 
 

SECTION: Tissue Digestion with CAP
15.2. Triturate with 1000 µL wide-bore tips to release leftover cells.
SECTION: Tissue Digestion with CAP
15.3. Pulse centrifuge (520 x g, 4 °C) to pellet tissue fragments, then collect as much supernatant as possible into the tube containing single cells from step 11. Do not disturb the non-dissociated tissue fragments at the base of the tube.
SECTION: Tissue Digestion with CAP
16. Centrifuge the collected cell suspension 800 x g, 8 min, 4 °C, then discard the supernatant. 

SECTION: Tissue Digestion with CAP
17. Resuspend the cell pellet with 2 mL , incubate on ice for 2-5 minutes to remove erythrocytes.
 .

SECTION: Tissue Digestion with CAP
18. Add 45 mL to stop ACK Lysis buffer reaction, then collect cells by centrifugation 800 x g, 5 min, 4 °C .

SECTION: Tissue Digestion with CAP
19. Resuspend the cell pellet in 2 mL or   
 



SECTION: Tissue Digestion with CAP
20. Filter the cell suspension through into   to remove debris and non-dissociated tissue fragments. The cell suspension is ready for flow cytometry cell staining and sorting. 

Note: We recommend filtering the cells before sorting through a  
SECTION: Viable Cell Sorting
34. Sort viable cells (PI-/Calcein Violet+) onto 5 mL collection tube (LoBind Eppendorf 5ml tube) containing 300 µL. We target to collect at least 40,000 cells per sample.
 

SECTION: Viable Cell Sorting
22. Top up the tube with 0.04 % (v/v) up to 5 mL, then centrifuge 2000 rpm, 5 min, 4 °C  to collect cells. Remove supernatant carefully until left with around 30-50 µL.
SECTION: Viable Cell Sorting
23. Resuspend carefully with 200 µL tips. Count the number of cells and cell viability with Countess FLII or similar preferred cell counting methodology.
 

SECTION: Tissue Digestion with CAP
6. Place 60 mm2 dish on ice, then use sterile forceps to transfer tissue from   onto the dish.

SECTION: Tissue Digestion with CAP
15.1. Add 3 mL into gentleMACS C tube. Placed the tube on gentleMACS Dissociator and Run “37C_h_TDK3” protocol for 15 min. This protocol requires the temperature set to 37 °C .

SECTION: Further use of dissociated single-cell
25. Leftover single-cells from this protocol can be used to generate endometrium organoids. To generate organoids, we utilize the protocol made by Sheridan et al. (2020) grown in endometrium organoid culture media optimized by Boretto et al. (2019).

 

 

SECTION: Buffer and Enzymes Preparation
2. Prepare fresh digestion enzyme - Dispase solution: 
add 500 µL 
add 10 mL 
add 40 µL 

SECTION: Buffer and Enzymes Preparation
3. Prepare Buffer I (For Washing and Collection of Dissociated Cells)
DPBS
2 mM EDTA
2% BSA
15% FBS
SECTION: Buffer and Enzymes Preparation
4. Prepare Buffer II (For Collection of Sorted Cells)
Advanced DMEM/F12 (1X)
1X Glutamax 
10 mM HEPES
10% FBS 
1% BSA 
SECTION: Buffer and Enzymes Preparation
5. Prepare Coating Buffer (For coating tubes/tips) 
2.5% BSA (keep on ice).