Nov 08, 2022
Human endometrium and endometriosis tissue dissociation for single-cell RNA sequencing
- Yuliana Tan1,
- Diane Luo2,
- Suleyman Bozal2,
- Paul Robson3,
- Elise Courtois2
- 1University of Connecticut Health Centre and The Jackson Laboratory for Genomic Medicine;
- 2Single Cell Biology, The Jackson Laboratory for Genomic Medicine;
- 3The Jackson Laboratory for Genomic Medicine
Protocol Citation: Yuliana Tan, Diane Luo, Suleyman Bozal, Paul Robson, Elise Courtois 2022. Human endometrium and endometriosis tissue dissociation for single-cell RNA sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.bvy8n7zw
Manuscript citation:
Single cell analysis of endometriosis reveals a coordinated transcriptional program driving immunotolerance and angiogenesis across eutopic and ectopic tissues. Yuliana Tan, William F. Flynn, Santhosh Sivajothi, Diane Luo, Suleyman B. Bozal, Monica Davé, Anthony A. Luciano, Paul Robson, Danielle E. Luciano, Elise T. Courtois Nature Cell Biology (2022)
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 21, 2021
Last Modified: November 08, 2022
Protocol Integer ID: 50944
Keywords: endometriosis, single-cell RNA sequencing, tissue dissociation, endometrium, sample preparation
Funders Acknowledgement:
CDMRP DoD
Grant ID: W81XWH1910130
Disclaimer
Icon on this protocol was created with Biorender.com.
Abstract
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.
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.
CITATION
Materials
CONSUMABLES
- Aspen Surgical™ Bard-Parker™ Protected Disposable Scalpel No. 10Fisher ScientificCatalog #02-688-78
- tissue culture dish 60 x 15 mmFisher ScientificCatalog #08772B
- Nalgene Rapid-Flow Sterile Single Use Vacuum Filter UnitsThermo Fisher ScientificCatalog #568-0020
- Falcon 50mL Conical Centrifuge TubesFisher ScientificCatalog #14-959-49A
- Falcon 15 mL Polystyrene Conical TubeFisher ScientificCatalog #352095
- Protein LoBind® Tubes 5mLEppendorfCatalog #0030108302
- MACS SmartStrainers (70 µm)Miltenyi BiotecCatalog #130-098-462
- gentleMACS C-Tube Miltenyi BiotecCatalog #130-093-334
- Protein LoBind 5 mL Tube Fisher ScientificCatalog #14-282-304
- Falcon 5 mL Polystyrene Round-Bottom Tube with Cell-Strainer CapFisher ScientificCatalog #352235
- Low-retention, filtered tips (1000 µL, 1000 µL wide bore, 200 µL, 20 µL)
- Serological Pipettes (5 mL, 10 mL, 50 mL)
REAGENTS
- 1X Dulbecco’s Phosphate Buffered Saline (DPBS) Thermo Fisher ScientificCatalog #14190094
- MACS BSA Stock SolutionMiltenyi BiotecCatalog # 130-091-376
- Fetal Bovine Serum, certified, heat inactivated, United StatesThermo FisherCatalog #10082139
- EDTA (0.5 M, pH 8.0, nuclease-free)Thermo Fisher ScientificCatalog #AM9260G
- Protease from Bacillus Licheniformis SigmaCatalog #P5380 (Cold Active Protease)
- DNase I Solution (1 mg/mL)Stemcell TechnologiesCatalog #07900
- 1 M Calcium Chloride (CaCl2)Fisher ScientificCatalog #BP510
- 1 mg/mL Dispase
- Advanced DMEM/F-12Thermo FisherCatalog #12634010
- Glutamax (100x)Gibco - Thermo FischerCatalog #35050-061
- HEPES 1MThermo Fisher ScientificCatalog #15630106
- MACS Tissue Storage SolutionMiltenyi BiotechCatalog #130-100-008
- ACK Lysing BufferThermo Fisher ScientificCatalog #A1049201
- Propidium iodide staining solutionBD BiosciencesCatalog #556463
- CellTrace™ Calcein Violet, AM, for 405 nm excitation - Special PackagingThermo FisherCatalog #C34858
- Cell Staining BufferBioLegendCatalog #420201
10X GENOMICS REAGENTS
- Chromium Next GEM Chip G Kit 10x GenomicsCatalog #1000120
- Chromium Next GEM Single Cell 3-prime Kit v3.110x GenomicsCatalog #1000268
- Dual Index Kit TT Set A10x GenomicsCatalog #1000215
- Dynabeads MyOne Silane10x GenomicsCatalog #2000048
- 0.2 mL PCR 8 Strip Magnetic Separator 5 uL ~ 0.2 mL VolumePermagenCatalog #MSRLV08 (Optional)
CRITICAL EQUIPMENT
- Centrifuge with temperature control
- Cell counter (e.g., countess II FL, Thermo Fisher)
Equipment
gentleMACS
NAME
gentleMACS™ Octo Dissociator with Heaters
TYPE
Miltenyi Biotec
BRAND
130-096-427
SKU
LINK
Equipment
FACSAria Fusion
NAME
Flow cytometer
TYPE
BD
BRAND
Special order
SKU
Equipment
Chromium Controller
NAME
microfuidic based single-cell capture
TYPE
10x Genomics
BRAND
1000171
SKU
LINK
Protocol materials
Fetal Bovine Serum, certified, heat inactivated, United StatesThermo FisherCatalog #10082139
Materials
Chromium Next GEM Chip G Kit 10x GenomicsCatalog #1000120
Materials, Step 24
HEPES 1MThermo Fisher ScientificCatalog #15630106
Materials
tissue culture dish 60 x 15 mmFisher ScientificCatalog #08772B
Materials
MACS BSA Stock SolutionMiltenyi BiotecCatalog # 130-091-376
Materials
0.2 mL PCR 8 Strip Magnetic Separator 5 uL ~ 0.2 mL VolumePermagenCatalog #MSRLV08
Materials
Nalgene Rapid-Flow Sterile Single Use Vacuum Filter UnitsThermo Fisher ScientificCatalog #568-0020
Materials
Advanced DMEM/F-12Thermo FisherCatalog #12634010
Materials
gentleMACS C-Tube Miltenyi BiotecCatalog #130-093-334
Materials, Step 9
Protein LoBind 5 mL Tube Fisher ScientificCatalog #14-282-304
Materials, Step 20
CellTrace™ Calcein Violet, AM, for 405 nm excitation - Special PackagingThermo FisherCatalog #C34858
Materials, Step 21
MACS Tissue Storage SolutionMiltenyi BiotecCatalog #130-100-008
Materials, Step 6
Propidium iodide staining solutionBecton Dickinson (BD)Catalog #556463
Materials, Step 21
Chromium Next GEM Single Cell 3-prime Kit v3.110x GenomicsCatalog #1000268
Materials, Step 24
ACK Lysing BufferThermo Fisher ScientificCatalog #A1049201
Materials, Step 17
1 M Calcium Chloride (CaCl2)Fisher ScientificCatalog #BP510
Materials
Aspen Surgical™ Bard-Parker™ Protected Disposable Scalpel No. 10Fisher ScientificCatalog #02-688-78
Materials
DNase I Solution (1 mg/mL)STEMCELL Technologies Inc.Catalog #07900
Materials
MACS SmartStrainers (70 µm)Miltenyi BiotecCatalog #130-098-462
Materials, Step 20
Protease from Bacillus Licheniformis Merck MilliporeSigma (Sigma-Aldrich)Catalog #P5380
Materials
Falcon 15 mL Polystyrene Conical TubeFisher ScientificCatalog #352095
Materials
Dual Index Kit TT Set A10x GenomicsCatalog #1000215
Materials
Falcon 5 mL Polystyrene Round-Bottom Tube with Cell-Strainer CapFisher ScientificCatalog #352235
Materials, Step 20
EDTA (0.5 M, pH 8.0, nuclease-free)Thermo Fisher ScientificCatalog #AM9260G
Materials
Protein LoBind® Tubes 5mLEppendorfCatalog #0030108302
Materials
1X Dulbecco’s Phosphate Buffered Saline (DPBS) Thermo Fisher ScientificCatalog #14190094
Materials
Dynabeads MyOne Silane10x GenomicsCatalog #2000048
Materials
Glutamax (100x)Gibco - Thermo FischerCatalog #35050-061
Materials
Falcon 50mL Conical Centrifuge TubesFisher ScientificCatalog #14-959-49A
Materials
Cell Staining BufferBioLegendCatalog #420201
Materials, Step 19
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).
Note
IMPORTANT:
Keep the cells cold (on ice) at all times, except when specifically mentioned in the protocol.
Note
Alternative to cold water bath:
- Fill ice bucket up to 3/4 of the volume, then top off with distilled water.
- Let the ice sit on bench for few minutes and measure the temperature.
Buffer and Enzymes Preparation
Buffer and Enzymes Preparation
30m
30m
Prepare fresh digestion enzyme - Cold Active Protease (CAP) solution:
| Reagents | Stock conc. | Final conc. | Vol. | |
| Cold Active Protease | 100 mg/mL | 10 mg/mL | 0.2 mL | |
| DPBS | 1X | 1X | 1.8 mL | |
| CaCl | 1M | 5 mM | 60 uL | |
| DNAseI | 1mg/mL | 8.3 uL |
Abbreviation:
conc. = concentration
vol = volume
Prepare fresh digestion enzyme - Dispase solution:
- add 500 µL Dispase (100 mg/mL)
- add 10 mL DPBS (1X)
- add 40 µL DNAseI (1 mg/mL)
Prepare Buffer I (For Washing and Collection of Dissociated Cells)
- DPBS
- 2 mM EDTA
- 2% BSA
- 15% FBS
Prepare Buffer II (For Collection of Sorted Cells)
- Advanced DMEM/F12 (1X)
- 1X Glutamax
- 10 mM HEPES
- 10% FBS
- 1% BSA
Prepare Coating Buffer (For coating tubes/tips)
- 2.5% BSA (keep On ice ).
Tissue Digestion with CAP
Tissue Digestion with CAP
Place 60 mm2 dish On ice , then use sterile forceps to transfer tissue from MACS Tissue Storage SolutionMiltenyi BiotecCatalog #130-100-008 onto the dish.
Mince the tissue to 1 mm2 cube with sterile scalpel.
5m
Add 2 mL CAP solution into the dish.
1m
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 gentleMACS C-Tube Miltenyi BiotecCatalog #130-093-334 with 2.5 % (v/v) BSA solution before use)
15m
Triturate the mixture with 1000 µL wide-bore tips for 00:00:15 every 00:02:00 to ensure homogenization and dissociation.
Note
We recommend the use of 1000 µL wide-bore tips for large tissue chucks and ensuring good homogenization and dissociation. However, most eutopic endometrium consists of mucuos layer and may be better dissociated with standard 1000 µL tips.
2m 15s
Place tube on gentleMACS Dissociator and run “mouse_brain_3” protocol. Repeat twice (2x).
Equipment
gentleMACS
NAME
gentleMACS™ Octo Dissociator with Heaters
TYPE
Miltenyi Biotec
BRAND
130-096-427
SKU
LINK
2m
When the run is finished, place the tube in cold water bath and let tissue settle down for 00:00:30 to 00:01:00
1m 30s
Fibrous or high fat content tissue19 steps
Fibrous or high fat content tissue will result in cloudy supernatant and tissue will not properly settled down.
To reduce possibility of collecting undissociated tissue fragments, do short centrifugations 520 x g, 4°C, 00:00:05 .
Follow this step on every single-cell supernatant collection.
Transfer 1-1.5 mL supernatant containing dissociated single-cells to 50 mL conical tube containing 30 mL Buffer I .
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.
1m
Add another freshly made 2 mL CAP solution into gentleMACS C Tubes containing the remaining non-dissociated tissue fragments. Incubate in cold water bath for 00:20:00 to 00:40:00 .
1h
Triturate the mixture with 1000 µL wide-bore tips for 00:00:15 every 00:05:00 incubation to ensure homogenization and dissociation.
Place tube on gentleMACS Dissociator and run “mouse_brain_3” protocol every 00:15:00 incubation
Equipment
gentleMACS™ Dissociator
NAME
tissue dissociator
TYPE
Miltenyi Biotec
BRAND
130-093-235
SKU
LINK
8 tubes, etc..
SPECIFICATIONS
Repeat go to step #11 to Step 8 . Transfer the supernatant to the 50 ml conical tube containing single cells from step 8.
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, continue to Step 12 .
- otherwise, follow the step below.
Note
Do this step only if tissue is not completely dissociated.
15m
Add 3 mL Dispase solution into gentleMACS C tube. Placed the tube on gentleMACS Dissociator and Run “37C_h_TDK3” protocol for 00:15:00 . This protocol requires the temperature set to 37 °C .
15m
Triturate with 1000 µL wide-bore tips to release leftover cells.
2m
Pulse centrifuge (520 x g, 4°C , 10 - 15 seconds ) 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.
30s
Centrifuge the collected cell suspension 800 x g, 4°C, 00:08:00 , then discard the supernatant.
8m
Resuspend the cell pellet with 2 mL ACK Lysing buffer , incubate on ice for 2-5 minutes to remove erythrocytes.
ACK Lysing BufferMiltenyi BiotecCatalog #A1049201 .
5m
Add 45 mL Buffer I to stop ACK Lysis buffer reaction, then collect cells by centrifugation 800 x g, 4°C, 00:05:00 .
5m
Resuspend the cell pellet in 2 mL Buffer II or Cell Staining BufferMiltenyi BiotecCatalog #420201
Note
Observe the cell suspension under the microscope. If aggregates of cells are observed, pellet the cells, remove the supernatant and continue dissociation with 500 µL TrypLE Express Enzyme for 00:02:00 to 00:03:00 , at 37 °C . Add 4.5 mL Buffer II to stop the reaction, pellet the cells and resuspend in either Buffer II or Cell Staining Buffer.
TrypLE™ Express EnzymeThermo Fisher ScientificCatalog #12604013
1m
Filter the cell suspension throughMACS SmartStrainers (70 µm)Miltenyi BiotecCatalog #130-098-462 into Protein LoBind 5 mL Tube Miltenyi BiotecCatalog #14-282-304 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 aFalcon 5 mL Polystyrene Round-Bottom Tube with Cell-Strainer CapMiltenyi BiotecCatalog #352235
Viable Cell Sorting
Viable Cell Sorting
2h
2h
Stain cells with viability dye (Propidium Iodide and Calcein Violet) following manufacturer's protocol, incubate 00:20:00 on ice .
- Propidium iodide staining solutionMiltenyi BiotecCatalog #556463
- CellTrace™ Calcein Violet, AM, for 405 nm excitation - Special PackagingMiltenyi BiotecCatalog #C34858
20m
Top up the tube with 0.04 % (v/v) BSA solution up to 5 mL , then centrifuge 2000 rpm, 4°C, 00:05:00 to collect cells. Remove supernatant carefully until left with around 30-50 µL of supernatant .
5m
Resuspend carefully with 200 µL tips. Count the number of cells and cell viability with Countess FLII or similar preferred cell counting methodology.
Equipment
Countess II
NAME
AMQAX1000
TYPE
Life Technologies/Invitrogen
BRAND
None
SKU
LINK
Setup for Cell Capture on 10x Chromium
Setup for Cell Capture on 10x Chromium
30m
30m
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).
Equipment
Chromium Controller
NAME
microfuidic based single-cell capture
TYPE
10x Genomics
BRAND
1000171
SKU
LINK
- Chromium Next GEM Single Cell 3-prime Kit v3.1Miltenyi BiotecCatalog #1000268
- Chromium Next GEM Chip G Kit Miltenyi BiotecCatalog #1000120 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.
Further use of dissociated single-cell
Further use of dissociated single-cell
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).
CITATION
CITATION
Citations
Adam M, Potter AS, Potter SS. Psychrophilic proteases dramatically reduce single-cell RNA-seq artifacts: a molecular atlas of kidney development.
https://doi.org/10.1242/dev.151142Step 25
Sheridan MA, Fernando RC, Gardner L, Hollinshead MS, Burton GJ, Moffett A, Turco MY. Establishment and differentiation of long-term trophoblast organoid cultures from the human placenta.
https://doi.org/10.1038/s41596-020-0381-xStep 25
Boretto M, Maenhoudt N, Luo X, Hennes A, Boeckx B, Bui B, Heremans R, Perneel L, Kobayashi H, Van Zundert I, Brems H, Cox B, Ferrante M, Uji-I H, Koh KP, D'Hooghe T, Vanhie A, Vergote I, Meuleman C, Tomassetti C, Lambrechts D, Vriens J, Timmerman D, Vankelecom H. Patient-derived organoids from endometrial disease capture clinical heterogeneity and are amenable to drug screening.
https://doi.org/10.1038/s41556-019-0360-z