Jul 31, 2025
Human breast cancer patient tissue dissociation for single-cell RNA sequencing
- Furkan Ozmen1,
- Tugba Y Ozmen1
- 1OHSU
Protocol Citation: Furkan Ozmen, Tugba Y Ozmen 2025. Human breast cancer patient tissue dissociation for single-cell RNA sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov11n7pvr2/v1
Manuscript citation:
Single-cell RNA sequencing reveals different cellular states in malignant cells and the tumor microenvironment in primary and metastatic ER-positive breast cancer. Furkan Ozmen, Tugba Y. Ozmen, Aysegul Ors, Mahnaz Janghorban, Matthew J. Rames, Xi Li, Aaron Reid Doe, Fariba Behbod, Gordon B. Mills, Hisham Mohammed NPJ Breast Cancer (2025)
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: July 31, 2025
Last Modified: July 31, 2025
Protocol Integer ID: 223821
Keywords: single-cell RNA sequencing, tissue dissociation, sample preparation, sequencing tissue dissociation, processing fresh human breast cancer tissue, human breast cancer patient tissue dissociation, transcriptomic integrity, fresh human breast cancer tissue, cell rna, maximizing viable cell recovery, enzymatic digestion with mechanical dissociation, viable cell recovery, reliable downstream analysis of the tumor microenvironment, strong transcript capture, gentle dissociation of solid tumor, tumor microenvironment, cell suspension, combining enzymatic digestion, preserving cell viability, sequencing efficient, cell viability, cell resolution
Abstract
Efficient and gentle dissociation of solid tumors is essential for preserving cell viability and transcriptomic integrity in single-cell RNA sequencing workflows. This protocol outlines an optimized approach for processing fresh human breast cancer tissue, designed to minimize cell stress while maximizing viable cell recovery. By combining enzymatic digestion with mechanical dissociation, the method yields high-quality single-cell suspensions with consistently high viability and strong transcript capture, enabling reliable downstream analysis of the tumor microenvironment at single-cell resolution.
Guidelines
This protocol is optimized for the dissociation of fresh human breast cancer tissue to generate high-quality single-cell suspensions suitable for RNA sequencing. When performed promptly and under proper conditions, the method yields high cell viability and robust transcript capture, resulting in high UMI and gene counts per cell.
Breast tumor specimens are collected fresh from the operating room and immediately submerged in MACS Tissue Storage Solution to preserve cell integrity during transport on ice. Timely processing upon arrival in the laboratory is critical to maintaining viability and ensuring high-quality single-cell RNA sequencing results.
Manuscript citation
Please remember to cite the following publication along with this protocol
Single-cell RNA sequencing reveals different cellular states in malignant cells and the tumor microenvironment in primary and metastatic ER-positive breast cancer. Furkan Ozmen, Tugba Y. Ozmen, Aysegul Ors, Mahnaz Janghorban, Matthew J. Rames, Xi Li, Aaron Reid Doe, Fariba Behbod, Gordon B. Mills, Hisham Mohammed NPJ Breast Cancer (2025)
Materials
CONSUMABLES
• Falcon 5 mL Polystyrene Round-Bottom Tube with Cell-Strainer Cap
Fisher Scientific — Catalog #352235
• Protein LoBind‱ Tubes 5 mL
Eppendorf — Catalog #0030108302
Fisher Scientific — Catalog #14-282-304
• Falcon 15 mL Polystyrene Conical Tube
Fisher Scientific — Catalog #352095
• Falcon 50 mL Conical Centrifuge Tubes
Fisher Scientific — Catalog #14-959-49A
• Nalgene Rapid-Flow Sterile Single Use Vacuum Filter Units
Thermo Fisher Scientific — Catalog #568-0020
• Tissue Culture Dish 60 x 15 mm / Petri Dish
Fisher Scientific — Catalog #08772B
• Micro Dissecting Scissors, 3.5” Straight Sharp
Roboz — Catalog #RS-5910
• Tweezer (standard sterile forceps)
• gentleMACS C-Tube
Miltenyi Biotec — Catalog #130-093-334
• MACS SmartStrainers (70µm)
Miltenyi Biotec — Catalog #130-098-462
• 40µm Cell Strainer
Fisher Scientific — Catalog #07-201-430
• FACS Tubes with Cell Strainer Cap
Fisher Scientific — Catalog #08-771-23
• 5 mL Eppendorf Tubes
Fisher Scientific — Catalog #14-282-301
• Aspen Surgical‱ Bard-Parker‱ Protected Disposable Scalpel No. 10
Fisher Scientific — Catalog #02-688-78
• 1 mL and 5 mL syringe plungers (for mashing tissue on strainers)
• Low-retention, filtered pipette tips
(1000 µL, 1000 µL wide bore, 200 µL, 20 µL)
• Serological Pipettes
(5 mL, 10 mL, 50 mL)
• Kimwipes
⸻
REAGENTS
• Phenol Red-Free RPMI, Thermo Fisher cat# 11835030
• 1X Dulbecco’s Phosphate Buffered Saline (DPBS), no calcium, no magnesium, Thermo Fisher Scientific — Catalog #14190094
• Fetal Bovine Serum (FBS), Certified, Heat Inactivated, U.S. Origin Thermo Fisher — Catalog #10082139
• Trypan Blue Dye, Gibco cat#15250061
• Y-27632 (ROCK Inhibitor), Selleck Chemicals — Catalog #S1049 (Resuspend in water to 10mM (10µM final), aliquot, and store at −80°C or −20°C short term.)
• gentleMACS Human Tumor Dissociation Enzymes, Miltenyi Biotec — Catalog #130-095-929
⸻
MAGNETIC REMOVAL REAGENTS
• EasySep Dead Cell Removal (Annexin V) Kit, STEMCELL Technologies — Catalog #17899
• EasySep RBC Depletion Reagent, STEMCELL Technologies — Catalog #18170
• EasySep Magnet, STEMCELL Technologies — Catalog #18000
• Biotin Selection Cocktail
• RapidSpheres (vortex before use)
⸻
10X GENOMICS REAGENTS
• Chromium Next GEM Chip G Kit
10x Genomics — Catalog #1000120
• Chromium Next GEM Single Cell 3′ Kit v3.1
10x Genomics — Catalog #1000268
• Dual Index Kit TT Set A
10x Genomics — Catalog #1000215
• Dynabeads MyOne Silane
10x Genomics — Catalog #2000048
• 0.2 mL PCR 8-Strip Magnetic Separator (optional)
Permagen — Catalog #MSRLV08
⸻
CRITICAL EQUIPMENT
• gentleMACS Dissociator
• Centrifuge with temperature control
• Cell counter (e.g., Countess, Thermo Fisher)
• Benchtop incubator with rocker
• Standard pipettes and multichannel pipettes
• 10X Genomics Chromium Controller
Protocol materials
Phenol red free RPMI, +/- 5% CS-FBS (charcoal stripped)Gibco - Thermo Fisher ScientificCatalog # A338210
Dulbeccos Phosphate-Buffered Saline (DPBS), No Calcium, No MagnesiumGibco - Thermo Fisher ScientificCatalog #14190144
EasySep Dead Cell Removal (Annexin V) Kit (17899)STEMCELL Technologies Inc.Catalog #17899
Y-27632 (ROCK inhibitor) (Selleck Chemicals Catalog # S1049)SelleckchemCatalog #S1049
gentleMACs human dissociation enzymes (130-095-929)Miltenyi BiotecCatalog #130-095-929
EasySep RBC Depletion Reagent (18170)STEMCELL Technologies Inc.Catalog #18170
EasySep Magnet (18000)STEMCELL Technologies Inc.Catalog #18000
Chromium Next GEM Single Cell 3-prime Kit v3.110x GenomicsCatalog #1000268
Chromium Next GEM Chip G Kit 10x GenomicsCatalog #1000120
Troubleshooting
Before start
Before You Start
This protocol is optimized for single-cell isolation from fresh human tumor biopsy cores using 10x Genomics 3’ v3.1 chemistry. Before beginning, ensure that:
Sample integrity is maintained by keeping tissue on ice at all times before dissociation.
Sterile technique is used throughout to prevent contamination and ensure sample quality.
All necessary sterile consumables and reagents prepared in advance.
A ROCK inhibitor stock (10 mM Y-27632) is pre-aliquoted and stored at −80°C.
Pre-aliquated dissociation enzymes and tubes are thawed.
Dissociation media is prepared fresh just prior to the dissociation step.
All centrifuges and incubators are set to appropriate temperatures (e.g., 4°C for centrifuge, 37°C for incubation).
The EasySep magnet and dead cell/RBC removal reagents, sterile scissors/tweezer sets are readily accessible and brought to room temperature as required.
Cell counter (Countess) is calibrated, clean, and functional.
Note
‱ Note: This protocol is optimized for small biopsy cores (≤2 mm). For larger chunks or solid tumors, dissociation time and filtering steps may require adjustments. Refer to notes throughout the procedure for alternate handling of large specimens.
Single cell isolation from fresh human tumor biopsy for 10X scRNA seq
This protocol is optimized for small biopsy cores. Adjust for big tumor chunks.
Reagents:
Phenol red free RPMI, +/- 5% CS-FBS (charcoal stripped)Gibco - Thermo Fisher ScientificCatalog # A338210
Dulbeccos Phosphate-Buffered Saline (DPBS), No Calcium, No MagnesiumGibco - Thermo Fisher ScientificCatalog #14190144
gentleMACs human dissociation enzymes (130-095-929)Miltenyi BiotecCatalog #130-095-929
EasySep Dead Cell Removal (Annexin V) Kit (17899)STEMCELL Technologies Inc.Catalog #17899
Y-27632 (ROCK inhibitor) (Selleck Chemicals Catalog # S1049)SelleckchemCatalog #S1049
EasySep RBC Depletion Reagent (18170)STEMCELL Technologies Inc.Catalog #18170
EasySep Magnet (18000)STEMCELL Technologies Inc.Catalog #18000
Chromium Next GEM Single Cell 3-prime Kit v3.110x GenomicsCatalog #1000268
Chromium Next GEM Chip G Kit 10x GenomicsCatalog #1000120
Equipments
Equipment
5ml Eppendorf Tubes
NAME
5ml Eppendorf Tubes
BRAND
14-282-301
SKU
Equipment
Falcon Round-Bottom (FACS) Tubes with Cell Strainer Cap (08-771-23) or 40μm cell strainer (07-201-430)
NAME
Falcon Round-Bottom (FACS) Tubes with Cell Straine
BRAND
08-771-23
SKU
Equipment
Falcon™ Standard Tissue Culture Dishes
NAME
Falcon™ Standard Tissue Culture Dishes
BRAND
08-772E
SKU
Equipment
new equipment
NAME
Biology Tweezer 115 mm, Ideal Tak
BRAND
7SG.CX.0
SKU
Biology Tweezer 115 mm (Ideal Tak Chiasso, Switzerland). Manufacturer Part Number: 7SG.CX.0
SPECIFICATIONS
Equipment
Micro Dissecting Scissors 3.5 Straight Sharp (RS-5910)
NAME
Roboz Surgical Store
BRAND
RS-5910
SKU
Equipment
Tube C (130-093-237)
NAME
GentleMacs
BRAND
130-093-237
SKU
Equipment
GentleMACS™ Octo Dissociator with Heaters
NAME
Dissociator with Heaters
TYPE
Miltenyi Biotec
BRAND
130-134-029
SKU
LINK
Equipment
Chromium Controller
NAME
microfuidic based single-cell capture
TYPE
10x Genomics
BRAND
1000171
SKU
LINK
Tumor dissociation:
Keep sample on ice until ready for dissociation.
Rinse the tissue in DPBS in a sterile plastic petri dish.
Add 2ml of phenol red-free RPMI to a Tube C with the cocktail of enzymes H+A+R plus Rock inhibitor (final 10uM).
While holding the Tube C tilted and maintaining media at the bottom of the tube, mince cells with a sharp scissor on the neck of the Tube C. Close the cap and mix well gently.
Run h_tumor_02 program on the gentleMACs dissociator. (for very hard tumors use h_tumor_01). Spin for short (5s) to collect media+cell at the bottom of the tube.
Incubate at 37°C on a bench incubator with rocker, gently rocking the media (speed 190) for 20-25 min. (Place the tube at a 45-degree angle while rocking).
After 20-25 min, gently pipette the tissue (4-5 times). For most biopsy cores, they will be completely dissociated after 20-25 min and with gentle pipetting. If there are still undissociated pieces after pipetting, run h_tumor_02 program on the gentleMACs dissociator. If still see big chunks, incubate for another 5-10 minutes.
For biopsy cores which are small additional incubation is not required. Mash the chunks on the strainer and they’ll dissociate and pass through the strainer.
Filter the dissociated cells+dissociation media through a 70μm cell strainer.
For Big and multiple cores: filter through a 40μm cell strainer in a 50ml tube. Mash the undissociated chunks with a 5ml syringe plunger.
For small cores, filter through a FACS Tubes with Cell Strainer Cap. Mash the undissociated chunks with a 1ml syringe plunger.
Wash the tube C with RPMI+2% CS-FBS to collect any residual cells and pass through the same strainer to wash the strainer as well. Transfer to a FACS tube if performing DEAD/RBC magnetic removal.
Spin at 4 degrees 300g for 7 min to collect cells
Decant the media gently without disturbing the pellet. While still holding the tube upside down dry the tube rim with a Kimwipe. Add DPBS+2%CS-FBS (total of ~200μl liquid in the tube with the leftover wash media).
Depending on tissue quality and appearance, dead cell and RBC removal can be performed in a combined or separate step. If the sample is particularly bloody, necrotic, or has low viability, it is recommended to perform sequential magnetic binding steps to improve removal efficiency and downstream cell quality.
Optional Isolate DEAD+RBC in a combined step (or separate if desired- Follow EasySep Protocols)
Add 100μL/mL of sample volume of Dead Cell Removal (Annexin V) Cocktail to sample (20μL for 200μL of sample).
Add 100μL/mL of sample volume of Biotin Selection Cocktail to sample.
Gently mix by tapping the tube, incubate @RT for 3 minutes
Vortex the RapidSpheres and add 100μL/mL of sample volume of RapidSpheres and mix gently.
Add DPBS+2%CS-FBS to top up the sample to 2.5ml. Mix by gently pipetting up and down 2-3 times
Add 100μL/mL of original sample volume of RBC Depletion Reagent to the 2.5ml mix.
Place the tube containing the DEAD cell removal+ RBC Depletion reagent in a magnet and incubate for 5min.
Pick up the magnet, and in one continuous motion invert the magnet and tube, pouring the cell suspension into a new 5ml tube.
Cell Counting
Count 3 times on the countess and average the count.
Adjust volume so that there is 700-1,200 cells/μl for 10X Genomics 3’ v3.1 protocol.
If there are still RBC or dead cells after the spin and count, repeat DEAD+RBCC removal one more time and incubate in the magnet for 5min to increase the purity.
Cell Capture on 10x Chromium
Load 20,000 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).
Protocol references
Single-cell RNA sequencing reveals different cellular states in malignant cells and the tumor microenvironment in primary and metastatic ER-positive breast cancer. Furkan Ozmen, Tugba Y. Ozmen, Aysegul Ors, Mahnaz Janghorban, Matthew J. Rames, Xi Li, Aaron Reid Doe, Fariba Behbod, Gordon B. Mills, Hisham Mohammed NPJ Breast Cancer (2025)
Chromium Next GEM Single Cell 3-prime kit v3.1, manufacturer's protocol (CG000315 Rev A).