Mar 19, 2025
Nuclei Isolation from Human Frozen Endometrium with Sample Multiplexing for 10X Genomics Multiome (ATAC + Gene Expression) Assay
- Shirin Issa Bhaloo1,
- Jade EB Carter1
- 1New York Genome Center
- New York Genome Center
Protocol Citation: Shirin Issa Bhaloo, Jade EB Carter 2025. Nuclei Isolation from Human Frozen Endometrium with Sample Multiplexing for 10X Genomics Multiome (ATAC + Gene Expression) Assay. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov12jq2gr2/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
We use this protocol and it's working
Created: March 18, 2025
Last Modified: March 19, 2025
Protocol Integer ID: 124569
Keywords: Nuclei Isolation, Endometrium, Frozen Tissue, Multiome, Sample Multiplexing, Nuclei Hashing
Funders Acknowledgements:
Chan Zuckerberg Initiative
Grant ID: DAF2021-239862
Abstract
This protocol was developed to enable the isolation of nuclei from human frozen endometrium suitable for the 10X Genomics Multiome (ATAC + Gene Expression) assay. Nuclei are prepared by mechanical homogenization of the tissue under mild cell lysis conditions, followed by washing steps. To reduce technical variability and costs, samples are multiplexed by hashing the nuclei of each sample with a unique oligonucleotide-tagged antibody against the nuclear pore complex. Hashed nuclei samples are pooled together at equal ratio and the resulting pool is permeabilized. Sample multiplexing also allows super-loading, however, it does not decrease the cost of sequencing. Prior to loading onto the Chromium X, the nuclei pool is transposed as per the Multiome (10X Genomics) protocol.
Guidelines
- This protocol has been optimized for non-tumor human frozen endometrium tissue. The human endometrium tissue is highly heterogeneous and therefore NP-40 concentration may need to be adapted to the tissue/sample condition. Whenever possible, performing a quick test on the tissue is very important to ensure good nuclei integrity. For the majority of samples tested with this protocol, 0.067% NP-40 was the optimal concentration.
- For a different tissue type, it is recommended to perform a trial on test samples to adjust parameters such as mincing time, lysis agent concentration, lysis time, mechanical homogenization method, and assess hashing feasibility.
- Always inspect the nuclei integrity under the microscope. Nuclei with good integrity are intact and their nucleoli may even be well visible. If nuclei clumping is observed, it is normally a sign of nuclei bursting due to over-lysis.
- Nuclei pooling takes place just before the permeabilization step and after hashing each sample independently with a unique HTO.
- In this protocol, a nuclei pool comprises 2 million nuclei corresponding to 0.5 million nuclei from each of the 4 hashed samples pooled together.
- Multiplexing a maximum of 4 samples is recommended. Nuclei hashing of more than 4 samples for a Multiome assay may result in less than 5000 nuclei recovered per sample after Seurat analysis.
Materials
REAGENTS AND CONSUMABLES
| Item | Vendor | Catalog # | |
| 1.5mL DNA LoBind Tubes | Eppendorf | 022431021 | |
| 2mL DNA LoBind Tubes | Eppendorf | 022431048 | |
| Acridine Orange/Propidium Iodide Stain | Logos Biosystems | F23001 | |
| Bel-Art Flowmi Cell Strainers (40 µm) | Fisher Scientific | 14-100-150 | |
| Calcium Chloride | VWR | 97062-590 | |
| Cell Staining Buffer | BioLegend | 420201 | |
| CELLTRICS 20 µm filters | Fisher Scientific | NC9699018 | |
| Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Bundle, 16 rxns | 10X Genomics | 1000283 | |
| Digitonin (5%) | Invitrogen | BN2006 | |
| Disposable Scalpels, Sterile, Sklar | VWR | 82029-850 | |
| DTT Solution (1M) | Tribioscience | TBS5039 | |
| EZFlow Filter Unit, Foxx PES Membrane 0.22µm Pore Size, Sterile | Foxx Life Sciences | 371-2215-OEM | |
| Human TruStain FcX (Fc Receptor Blocking Solution) | BioLegend | 422302 | |
| KIMBLE Dounce tissue grinder set | Sigma-Aldrich | D8938-1SET | |
| LUNA-FX7 Automated Cell Counter | Logos Biosystems | L70001 | |
| MACS BSA Stock Solution | Miltenyi Biotec | 130-091-376 | |
| MACS SmartStrainers (30 µm) | Miltenyi Biotec | 130-098-458 | |
| Magnesium Acetate Tetrahydrate | VWR | 97061-060 | |
| Magnesium Chloride Solution (1M) | Sigma-Aldrich | M1028 | |
| Molecular Grade Water (Nuclease-free Water) | G-Biosciences | 786-292 | |
| Nonidet P 40 Substitute (NP-40) | Sigma-Aldrich | 74385 | |
| PhotonSlide, 50 Slides | Logos Biosystems | L12005 | |
| Pipette Tips RT LTS 1000µL FLW 768A/8 (Wide bore tips) | Mettler Toledo | 30389218 | |
| Polysorbate 20 (Tween-20) | VWR | 97062-332 | |
| Protector RNase Inhibitor | Roche | 3335399001 | |
| Recombinant RNase Inhibitor | Takara | 2313B | |
| RNaseZap RNase Decontamination Solution | Thermo Fisher Scientific | AM9780 | |
| Sodium Chloride Solution (5M) | Sigma-Aldrich | 71386-1L | |
| SP Bel-Art Disposable Polypropylene Pestles | VWR | 47750-354 | |
| Sucrose | VWR | 470302-810 | |
| TotalSeq-A anti-Nuclear Pore Complex Proteins Hashtag Antibodies (HTO) | BioLegend | Varied | |
| Trizma hydrochloride solution (pH 7.4, 1M) | Sigma-Aldrich | T2194-1L | |
| Trypan Blue Solution, 0.4% | Thermo Fisher Scientific | 15250061 |
STOCK BUFFERS
- All stock buffers should be kept at 4°C for a maximum of 6 months.
- To all buffers add only right before use: 1mM DTT and 1U/µL RNase Inhibitor.
- Transposition mix should only be prepared when ready for the transposition step.
- Prepare an NP-40 stock solution of 10%.
- The volumes indicated for the stock buffers are sufficient for 4 samples or 1 pool.
Homogenization Suspension Buffer for Lysis (HSB-1) – 15 mL
| Reagents | Volume (µL) | Final Concentration | |
| Sucrose (1M) | 4800 | 320mM | |
| Tris-HCl pH 7.4 (1M) | 150 | 10mM | |
| Calcium Chloride (1M) | 45 | 3mM | |
| Magnesium Acetate Tetrahydrate (1M) | 45 | 3mM | |
| DTT Solution (1M) | 15 | 1mM | |
| Protector RNase Inhibitor (40U/µL), Roche | 375 | 1U/µL | |
| Nuclease-free Water | 9570 | NA | |
| Total Volume | 15000 | NA |
Homogenization Suspension Buffer for Washes (HSB-2) – 25 mL
| Reagents | Volume (µL) | Final Concentration | |
| Sucrose (1M) | 8000 | 320mM | |
| Tris-HCl pH 7.4 (1M) | 250 | 10mM | |
| Calcium Chloride (1M) | 75 | 3mM | |
| Magnesium Acetate Tetrahydrate (1M) | 75 | 3mM | |
| DTT Solution (1M) | 25 | 1mM | |
| Recombinant Rnase Inhibitor (40U/µL), Takara | 625 | 1U/µL | |
| Nuclease-free Water | 15950 | NA | |
| Total Volume | 25000 | NA |
Note
- For tissue lysis steps use HSB-1 (Protector RNase Inhibitor, Roche).
- For quenching-wash steps use HSB-2 (Recombinant RNase Inhibitor, Takara).
- Filter the HSB buffers using a 0.22 µm PVDF filter unit.
Nuclei Staining Buffer for Incubation (NSB-1) – 2 mL
| Reagents | Volume (µL) | Final Concentration | |
| 10% Tween-20 | 2 | 0.01% | |
| Calcium Chloride (1M) | 1.8 | 0.9mM | |
| Magnesium Chloride (1M) | 1 | 0.5mM | |
| DTT Solution (1M) | 2 | 1mM | |
| Protector RNase Inhibitor (40U/µL), Roche | 50 | 1U/µL | |
| Cell Staining Buffer - BioLegend | 1943.2 | 1X | |
| Total Volume | 2000 | NA |
Nuclei Staining Buffer for Washes (NSB-2) – 20 mL
| Reagents | Volume (µL) | Final Concentration | |
| 10% Tween-20 | 20 | 0.01% | |
| Calcium Chloride (1M) | 18 | 0.9mM | |
| Magnesium Chloride (1M) | 10 | 0.5mM | |
| DTT Solution (1M) | 20 | 1mM | |
| Recombinant RNase Inhibitor (40U/µL), Takara | 500 | 1U/µL | |
| Cell Staining Buffer - BioLegend | 19432 | 1X | |
| Total Volume | 20000 | NA |
Note
- For hashtag antibody incubation step use NSB-1 (Protector RNase Inhibitor, Roche).
- For hashing washing steps use NSB-2 (Recombinant RNase Inhibitor, Takara).
Permeabilization Wash Buffer (PWB) – 3 mL
| Reagents | Volume (µL) | Final Concentration | |
| Tris-HCl pH 7.4 (1M) | 30 | 10mM | |
| Sodium Chloride (5M) | 6 | 10mM | |
| Magnesium Chloride (1M) | 9 | 3mM | |
| 10% Tween-20 | 3 | 0.01% | |
| MACS BSA Stock Solution | 300 | 1% | |
| DTT Solution (1M) | 3 | 1mM | |
| Protector RNase Inhibitor (40U/µL), Roche | 75 | 1U/uL | |
| Nuclease-free Water | 2574 | 1X | |
| Total Volume | 3000 | NA |
Permeabilization Digitonin Buffer (PDB) – 0.5 mL
| Reagents | Volume (µL) | Final Concentration | |
| 10% NP-40 | 0.75 | 0.015% | |
| 0.5% Digitonin | 1 | 0.001% | |
| PWB | 498.25 | 1X | |
| Total Volume | 500 | NA |
Note
- There is no need to add RNase Inhibitor in the PDB buffer as this was added already in PWB.
Nuclei Buffer (NB) – 1 mL
| Reagents | Volume (µL) | Final Concentration | |
| 20X Nuclei Buffer (10X Genomics) | 50 | 1X | |
| DTT Solution (1M) | 1 | 1mM | |
| Protector RNase Inhibitor (40U/µL), Roche | 25 | 1U/uL | |
| Nuclease-free Water | 924 | 1X | |
| Total Volume | 1000 | NA |
Before start
- All steps should be performed on ice or at 4°C.
- All reagents should be kept at 4°C.
- Pre-chill a swinging bucket centrifuge.
- Pre-chill all douncers, pestles, tubes, petri dishes, scalpels and forceps.
- Spray down all work surfaces with RNaseZap.
- Use disposable, RNase-free pipettes tips and tubes.
- Use wide bore tips where indicated.
- For each sample the following will be required: 1X glass douncer and respective glass pestle (only pestle A – loose pestle is needed), 1X petri dish, 1X scalpel, 1X forceps, 1X 15mL falcon tube fitted with a MACS SmartStrainer (30 µm), 3X 1.5mL Eppendorf DNA LoBind Tubes, 1X disposable polypropylene pestle.
- Remove samples from liquid nitrogen or -80°C storage and keep them on dry ice until use.
- Use 30-50mg of tissue per sample.
Tissue Homogenization and Nuclei Isolation
Tissue Homogenization and Nuclei Isolation
Place 30-50mg of frozen tissue on a pre-chilled petri dish and immediately add 150 µL of HSB-1 to cover the tissue.
On ice, quickly mince/chop the tissue into 2-3mm sized pieces.
Transfer the tissue pieces/HSB mix to a douncer. Rinse the petri dish with 450 µL of HSB-1 and transfer it to the same douncer. The total volume of HSB-1 in the douncer will be 600 µL .
Add 4 µL of 10% NP-40 (0.067% NP-40 final concentration) to the douncer and start the timer pre-set for 00:05:00 to initiate lysis incubation step.
Gently homogenize the sample using the loose pestle (pestle A) by stroking 15 times. Keep the douncer on ice at all times and avoid making bubbles.
Half way through the lysis incubation time, gently pipette mix 5 times the tissue lysate with a wide bore 1mL tip.
At the end of the 5 minute lysis incubation time, quench the lysate with 1 mL of HSB-1 and gently pipette mix 10 times with a wide bore 1mL tip.
Filter the homogenate through the 30 µm MACS SmartStrainer fitted into a 15mL falcon tube.
Rinse the douncer with 1 mL HSB-2 to recover any remaining tissue homogenate and filter it through the same MACS SmartStrainer.
Rinse the MACS SmartStrainer with an additional 2 mL of HSB-2 to force all nuclei to pass through the strainer.
Using forceps or a 1mL wide bore tip, quickly recover the bigger tissue pieces left on the strainer and transfer them to a 1.5mL Eppendorf tube. Immediately add 200 µL of HSB-1.
Very gently homogenize the tissue pieces 5 times with a disposable polypropylene pestle.
Rinse the disposable pestle with 300 µL of HSB-1 into the same Eppendorf tube containing the lysate.
Add 2 µL of 10% NP-40 (0.04% NP-40 final concentration) and start the timer pre-set for 00:03:00 . Gently pipette mix 10 times with a wide bore 1mL tip.
At the end of the 3 minute lysis incubation time, quench the lysate with 1 mL of HSB-1 and gently pipette mix 10 times with a 1mL wide bore tip.
Filter the homogenate through the same 30 µm MACS SmartStrainer fitted into a 15mL falcon tube from step 8.
Rinse the Eppendorf tube with 1 mL of HSB-2 to recover any remaining tissue homogenate and filter it through the MACS SmartStrainer.
Rinse the MACS SmartStrainer with an additional 2 mL of HSB-2.
Centrifuge the nuclei at 500 x g, 4°C, 00:05:00 .
Remove as much supernatant as possible without disturbing the nuclei pellet. Gently add 200 µL of NSB-1 without disturbing the pellet. Let it sit on ice for 00:05:00 .
After the incubation time, gently pipette mix at least 10 times to resuspend the nuclei pellet.
Count the nuclei using the AO/PI dye and an automatic cell counter (e.g. LUNA-FX7‱ Automated Cell Counter).
Nuclei Hashing and Pooling
Nuclei Hashing and Pooling
Transfer 1.5 million nuclei into a new 1.5mL Eppendorf tube. Bring the total volume up to 150 µL by adding appropriate amount of NSB-1 (Nuclei concentration: 1.5 million/150µL).
Note
- If volume is greater than 150µL, centrifuge at 500 x g, 4°C, 00:05:00 . Remove as much supernatant as possible. Gently resuspend the nuclei pellet with 150 µL of NSB-1. Ensure the pellet is fully resuspended and the nuclei suspension is homogenous.
- In the case where a lower number of nuclei are recovered, you may proceed with the hashing step by adjusting the quantity of HTO as described in step 25 below.
Add 5 µL of Human TruStain FcX blocking solution to each nuclei sample, pipette mix 5 times and incubate on ice for 00:10:00 .
After the blocking step, add to each sample 1.5 µg (3 µL ) of a unique hashtag antibody (Hashing concentration: 1.5 million nuclei/1.5µg HTO/150µL). Pipette mix 10 times.
Note
- Make note of which HTO was added to which sample. This is very important for downstream analysis and sample identification within a pool.
- Hashing concentration recommendations: 0.5-1 million nuclei/1μg HTO/100μL or 1.5 million nuclei/1.5µg HTO/150μL.
Incubate on ice for 00:30:00 .
Wash 1: After hashing incubation add 900 µL of NSB-2 to each sample.
Centrifuge at 500 x g, 4°C, 00:05:00 .
Carefully remove the supernatant, leaving behind just enough volume to cover the pellet.
Wash 2: Add 900 µL of NSB-2 to each sample. Gently pipette mix 5 times.
Centrifuge at 500 x g, 4°C, 00:05:00 .
Carefully remove the supernatant leaving behind just enough volume to cover the pellet.
Wash 3: Add 900 µL of NSB-2 to each sample. Gently pipette mix 5 times.
Centrifuge again at 500 x g, 4°C, 00:05:00 .
Carefully remove the supernatant without disturbing the pellet. Remove as much buffer as possible to reduce antibody carryover.
Resuspend the nuclei in 500 µL of NSB-2 by gently pipette mixing at least 10 times or until no aggregates are visible by eye.
Assess the nuclei suspension by checking for the presence of nuclei clumps or aggregates through trypan blue staining.
Note
- If aggregates/clumps are present, further pipette mix 5 times the nuclei suspension and filter through a CELLTRICS 20 µm filter fitted in a 1.5mL Eppendorf tube on ice. Rinse the filter with 500µL of NSB-2 to push all the nuclei through the filter.
Gently pipette mix the nuclei suspension and count the nuclei as described in step 22.
In a new 2mL Eppendorf tube add 0.5 million nuclei from each hashed sample (maximum of 4 samples or 2 million nuclei). Gently pipette mix 5 times.
Note
- It is very important to pool the nuclei from each sample at equal proportions. If one sample has a total of less than 0.5 million nuclei, use the same nuclei number for the other 3 samples.
Centrifuge at 500 x g, 4°C, 00:05:00 .
Nuclei Permeabilization and Transposition
Nuclei Permeabilization and Transposition
Remove as much supernatant as possible and gently add 500 µL of PWB without disturbing the pellet. Incubate on ice for 00:05:00 .
After the incubation time, very gently resuspend the nuclei pellet by pipette mixing 5 times.
Centrifuge the nuclei suspension at 500 x g, 4°C, 00:05:00 .
Remove completely the supernatant.
Carefully add 125 µL of PDB and start the timer pre-set to 00:02:00 . Gently pipette mix the nuclei 10 times and incubate the nuclei on ice.
After the incubation time, add 1 mL of PWB and pipette mix 3 times.
Centrifuge at 500 x g, 4°C, 00:05:00 .
Resuspend the nuclei in 125 µL of NB by gently pipette mixing at least 10 times.
Count the nuclei as described in step 22.
If nuclei aggregates or clumps are present, filter the nuclei pool through a 40 µm Flowmi Cell Strainer.
Count the nuclei again and aim for a concentration of 8-8.5 million nuclei/mL (4-4.25x104 nuclei in 5µL).
Note
- If nuclei concentration is more dilute than the required, proceed to centrifugation at 500 x g, 4°C, 00:05:00 to concentrate the nuclei suspension. Always consider a 50% loss of nuclei with each centrifugation post-permeabilization step.
Prepare the transposition mix as described in the 10X Genomics Multiome protocol. Aim to load 4-4.25x104 nuclei for a total volume of 5µL.
Proceed as per 10X Genomics Multiome protocol.
Protocol references
1. “Nuclei isolation from fresh and frozen brain tissue”, Schmutz et al., protocols.io, dx.doi.org/10.17504/protocols.io.bvhhn336.
2. “Protocol for nuclei isolation from fresh and frozen tissues for parallel snRNA-Seq and snATAC-Seq on 10x Chromiumium platform using the same nuclei preparation (UPDATED version) V.4”, Luciano G. Martelotto, protocols.io, dx.doi.org/10.17504/protocols.io.bdeni3de.
3. “Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Kits User Guide”, CG000338 Rev G, 10X Genomics