Feb 19, 2025

Public workspaceManual Uterine Tissue Dissociation for Multiome Analysis

DOI
https://dx.doi.org/10.17504/protocols.io.261gerpjol47/v1
  • Sarah Johnston1,
  • Kate O'Neill1,
  • Stephen Fisher1,
  • Junhyong Kim1
  • 1University of Pennsylvania
  • Human BioMolecular Atlas Program (HuBMAP) Method Development Community
    Tech. support email: [email protected]
Sarah A Johnston
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DOI: https://dx.doi.org/10.17504/protocols.io.261gerpjol47/v1
Protocol Citation: Sarah Johnston, Kate O'Neill, Stephen Fisher, Junhyong Kim 2025. Manual Uterine Tissue Dissociation for Multiome Analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.261gerpjol47/v1
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
This protocol is currently in use by our lab and continues to yield high-quality ATAC-seq and GEX libraries for 10X Genomics Multiome analysis.
Created: October 25, 2024
Last Modified: February 19, 2025
Protocol Integer ID: 110930
Keywords: ATACseq, RNAseq, Multiome, 10X Genomics, uterus, cervix, single nuclei, manual uterine tissue dissociation for multiome analysis, frozen uterine tissue, frozen uterine tissues in order, manual uterine tissue dissociation, high quality 10x multiome library, cervix tissue biopsy, multiome analysis, human biomolecular atlas program, freezing tissue, using fresh tissue, rna quality, tissue thawing, rna, slight decrease in rna quality, downstream molecular analysis, fresh tissue, tissue
Funders Acknowledgements:
NIH
Grant ID: U54HD104392
Abstract
This protocol was developed in accordance with the mission statement of the Human BioMolecular Atlas Program (HuBMAP) of the NIH, to create an open and global platform to map healthy cells in the human body. Protocols developed through HuBMAP can be used by researchers across the world to create Standard Operating Protocols (SOPs) for sample collection and processing.

This protocol describes dissociation of snap-frozen uterine tissues in order to isolate nuclei that can be used for downstream molecular analysis. This protocol can also be used on cell suspensions made from fresh tissue; freezing tissues leads to a slight decrease in RNA quality after tissue thawing and dissociation. If using fresh tissue, we recommend optimizing the lysis time. We use this protocol to generate high quality 10X Multiome libraries from snap-frozen uterine and cervix tissue biopsies.
Materials
Nuclease-free H2O
Reagent Nonidet P40 SubstituteMerck MilliporeSigma (Sigma-Aldrich)Catalog # 74385
Reagent Trizma Hydrochloride Solution pH 7.4 Merck MilliporeSigma (Sigma-Aldrich)Catalog #T2194
ReagentSodium chlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #59222C-1000ML
ReagentMagnesium chloride solution for molecular biology (1.00 M)Merck MilliporeSigma (Sigma-Aldrich)Catalog #M1028
ReagentTween-20Merck MilliporeSigma (Sigma-Aldrich)Catalog #P-7949
ReagentProtector RNase InhibitorMerck MilliporeSigma (Sigma-Aldrich)Catalog #3335399001
ReagentUltrapure BSAAmbionCatalog #AM2616
ReagentDTTMerck MilliporeSigma (Sigma-Aldrich)Catalog #43816-10ML
ReagentDigitonin (5%)Thermo FisherCatalog #BN2006
ReagentCell strainer 70um filterFalconCatalog #352350
ReagentBel-Art™ SP Scienceware™ Flowmi™ Cell Strainers for 1000 μL Pipette TipsFisher ScientificCatalog #14-100-150
ReagentTrypan BlueInvitrogen - Thermo FisherCatalog #T10282
ReagentLance Disposable Scalpel, Sterile Size 22, green handleElectron Microscopy SciencesCatalog #72054-48
Ice bucket
Dry ice
Wet ice

Lysis Dilution Buffer
10mM Tris-HLC, pH 7.4
10mM NaCl
3mM MgCl2
1% BSA
1mM DTT
1U/μL Protector RNase Inhibitor
Nuclease-free H2O

1X Lysis Buffer
10mM Tris-HLC, pH 7.4
10mM NaCl
3mM MgCl2
1% BSA
0.1% Tween-20
0.1% Nonidet NP-40 Substitute
0.01% Digitonin
1mM DTT
1U/μL Protector RNase Inhibitor
Nuclease-free H2O

0.1X Lysis Buffer
Dilute 1X Lysis buffer in Lysis Dilution Buffer to a final concentration of 0.1X.

Nuclei Wash Buffer
10mM Tris-HLC, pH 7.4
10mM NaCl
3mM MgCl2
1% BSA
0.1% Tween-20
1mM DTT
1U/μL Protector RNase Inhibitor
Nuclease-free H2O

Diluted Nuclei BufferReagent20X Nuclei buffer10x GenomicsCatalog #2000207
1X Nuclei Buffer (20X Nuclei buffer stock)
1mM DTT
1U/μL Protector RNase Inhibitor
Nuclease-free H2O
Equipment
C-Chip Disposable Hemocytometer, NI, 100 Tests
NAME
Hemocytometer
TYPE
Bulldog Bio
BRAND
102407-946
SKU
https://us.vwr.com/store/product/13648361/null
LINK
50 slides (2 tests/slide); chamber volume = 10uL
SPECIFICATIONS
Download Bulldogbio.Gif

Equipment
Falcon™ Plastic Disposable Transfer Pipets
NAME
Transfer pipet
TYPE
Falcon
BRAND
13-680-50
SKU
https://www.fishersci.com/shop/products/corning-plastic-disposable-transfer-pipets-2/1368050?searchHijack=true&searchTerm=357524&searchType=RAPID&matchedCatNo=357524
LINK
3mL volume with graduations
SPECIFICATIONS
Download Transfer Pipet.Jpg


Troubleshooting
Before start
Prepare all buffers fresh on wet ice the day of the experiment. All buffers must be maintained at 4°C.

Prepare a bucket with dry ice.

If using a ceramic mortar, place the mortar on dry ice to pre-chill. The mortar should be ice cold by the time you are ready to mince your sample.

If using a glass Dounce instead of a ceramic mortar, place the glass Dounce on wet ice to pre-chill. The Dounce should be ice-cold by the time you are ready to Dounce your sample.
Methods
59m 50s
Retrieve a snap frozen uterus/cervix sample and place it on dry ice.
5m
Using a scalpel and the pre-chilled mortar, cut a piece of tissue between 40-150mg, keeping the sample frozen.
1m
Using a disposable weigh boat, take the sample weight while keeping the sample frozen.
30s
In the pre-chilled mortar, mince the frozen tissue until it’s the size of (or a bit smaller) than grains of dry rice. 
Note
If the mortar is properly chilled, the tissue will remain frozen during the mincing process.


1m 30s
Using a mechanical tissue disruptor, such as a Drummel, homogenize the minced frozen uterine tissue in 400µL of 0.1X Lysis buffer.
Note
Adding more than 400µL of Lysis Buffer at this step may cause sample overflow during homogenization.

Note
In some cases using a mechanical tissue disrupter, such as a Drummel, may produce excessive debris.  In this case, you can homogenize the frozen tissue in 400µL 0.1X Lysis Buffer using a pre-chilled ice-cold Dounce homogenizer.  When using a Dounce, homogenize tissue using 12-20 stokes with the A (loose) pestle, followed by 15 strokes with the B (tight) pestle.
1m
Add 600µL of 0.1X Lysis Buffer to homogenized sample and pipet 2-5x with a 1000µL pipet tip or a wide-bore transfer pipet.
10s
Incubate for 5 minutes on ice.
5m
Pipette sample 10x using a 1000µL pipet tip or wide-bore transfer pipet.
10s
Incubate for 4 minutes on ice.
4m
For cervix samples, proceed to Step 11. For uterus samples, proceed to Step 12.
Note
Total lysis time for uterus samples is ~9 minutes.

Pipette sample 10x using a 1000µL pipet tip or wide-bore transfer pipet. Incubate for 10 min on ice.
Note
Total lysis time for cervix samples is ~20 minutes.

10m
Strain sample through a 70µm cell strainer.  The flow-through contains your nuclei suspension.
1m
Strain nuclei suspension through a 40µm Flowmi cell strainer.
1m
Add an equal volume of Nuclei Wash Buffer and pipette mix 3-5x using a 1000µL pipet tip or wide-bore transfer pipet.
30s
Centrifuge at 500 rcf for 5 minutes at 4°C.
5m
Discard the supernatant without disrupting the nuclei pellet.
20s
Add 500µL Nuclei Wash Buffer and gently pipette 5x to resuspend the nuclei pellet.
1m
Centrifuge at 500 rcf for 5 minutes at 4°C.
5m
Discard the supernatant without disrupting the nuclei pellet.
20s
Repeat Steps 17-19 for a total of 2 washes.
6m 20s
Add 500-1000µL Nuclei Wash Buffer.
20s
Determine the nuclei count using Trypan Blue exclusion.
Note
Quality control: If significant debris is observed, strain nuclei suspension through a 40µm Flowmi cell strainer; then, determine nuclei concentration using Trypan Blue exclusion.

Note
Quality control: There are acceptable levels of nuclei membrane blebbing, as represented in images A and B from Panel A in the 10X Genomics document CG000375. An acceptable nuclei suspension will contain <5% live cells (ideally no live cells), minimal to no clumping, minimal to no debris, and no large debris.
5m
Centrifuge at 500 rcf for 5 minutes at 4°C.
5m
Discard the supernatant without disrupting the nuclei pellet.
20s
Resuspend the nuclei pellet in ice-cold Diluted Nuclei Buffer to create your nuclei stock concentration. The volume is calculated based on your nuclei count obtained in step 22 and the appropriate concentration range as determined by your Targeted Nuclei Recovery.
Note
Quality control: for best results, the Targeted Nuclei Recovery should be between 5,000-6,000.
Note
To find the acceptable nuclei stock concentration ranges, refer to the "Nuclei Stock Concentration Table" on page 5 of the 10X Genomics protocol “Nuclei Isolation from Complex Tissues for Single Cell Multiome ATAC + Gene Expression Sequencing demonstration protocol.”

20s
Process the permeabilized nuclei with the 10X Genomics Multiomic ATACseq and RNAseq protocols described in “Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Kits User Guide.”
Protocol references
Acknowledgements
John Renz, Gift of Life Donor Program
Liming Pei, PhD, Children's Hospital of Philadelphia, TMC-CHOP
Po Hu, Children's Hospital of Philadelphia, University of Pennsylvania School of Medicine

All the organ donors and their families who so selflessly participate in our research.

This research is supported by the NIH Common Fund, through the Office of Strategic Coordination/Office of the NIH Director.