Dec 12, 2025
Nuclei isolation from frozen gonad tissue
- Maria del Carmen Sancho Serra or Carmen Sancho Serra1
- 1Wellcome Sanger Institute
- Vento-TormoTech. support email: [email protected]
Protocol Citation: Maria del Carmen Sancho Serra or Carmen Sancho Serra 2025. Nuclei isolation from frozen gonad tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvjxqewlk5/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: April 23, 2021
Last Modified: December 12, 2025
Protocol Integer ID: 49363
Keywords: nuclei, extraction, tissue, frozen, nuclei isolation from frozen gonad tissue, frozen gonad tissue, nuclei isolation, using dounce homogenizer, dounce homogenizer, ovary strip
Abstract
This protocol describes how to perform nuclei isolation from frozen gonad tissues using Dounce homogenizers. It can be used as well for ovary strips frozen in OCT
Guidelines
Human tissue and cells from primary samples may contain uncharacterized adventitious agents, including blood-borne viruses. No attempt will be made to culture these agents deliberately. Correct use of PPE will drastically reduce the risks.
Tissue collection for this protocol requires prior approval by the users' Institutional Ethics Board or equivalent ethics committee
Materials
SucroseMerck MilliporeSigma (Sigma-Aldrich)Catalog #S0389
KCl 2MCatalog #AM9640G
1M MgCl2Invitrogen - Thermo FisherCatalog #AM9530G
1M Tris buffer pH8.0InvitrogenCatalog #AM9855G
Triton X-100, 10% solution Merck MilliporeSigma (Sigma-Aldrich)Catalog #93443
1xPBS (RNase free) pH7.4Gibco - Thermo Fisher ScientificCatalog #10010-023
1M DL-Dithiothreitol solution (DTT)Merck MilliporeSigma (Sigma-Aldrich)Catalog #646563
cOmplete™ Protease Inhibitor CocktailMerck MilliporeSigma (Sigma-Aldrich)Catalog #11697498001
SUPERase• In™ RNase Inhibitor (20 U/μL)Thermo Fisher ScientificCatalog #cat# AM2694
UltraPure™ BSA (50 mg/mL)Thermo FisherCatalog #AM2618
RNasin(R) Plus RNase Inhibitor, 10,000uPromegaCatalog #N2615
KIMBLE Dounce tissue grinder set 7 mL completeMerck MilliporeSigma (Sigma-Aldrich)Catalog #D9063
Flowmi Cell StrainerMerck Millipore (EMD Millipore)Catalog #BAH136800040-50EA
Troubleshooting
Buffer preparation
Nuclei Isolation Buffer 1 (NIM1)
Can be made, filtered and stored at 4°C for up to 6 months
| A | B | C | D | |
| Start M/MW | Final M | Volume/amount | ||
| Sucrose | 342.3 g/mol | 0.25 | 4.279 g | |
| KCL | 2 mol/l | 0.025 | 625 µl | |
| MgCl2 | 1 mol/l | 0.005 | 250 µl | |
| Tris buffer pH8 | 1 mol/l | 0.01 | 500 µl | |
| Nuclease free water | - | - | 48.625 ml | |
| Total | 50 ml |
Nuclei Isolation Buffer 2 (NIM2)
To be made and used on the day of experiment, keep on ice.
- Pre-dilute 1M DTT: 5 ml 1 M DTT in 4.995 ml water.
- For 100x Protease inhibitor stock, dissolve 1 tablet in 500 ml nuclease free water. Stock solution can be stored at 2 to 8 °C for 1 to 2 weeks, or at least 12 weeks at -15 to -25 °C.
| A | B | C | D | |
| Start M/X | Final M | Volume/amount | ||
| DTT pre-made stock | 1 mol/l | 1 | 10 µl | |
| 100x protease inh. | 100x | 1 | 100 µl | |
| NIM1 | - | - | 9.89 ml | |
| Total | 10 ml |
Homogenization Buffer
To be made and used on the day of experiment, keep on ice. Do NOT vortex.
| A | B | C | D | |
| Start conc/% | Final conc/% | Volume/amount | ||
| RNaseIn Plus | 40 U/ µl | 0.4 | 100 µl | |
| Superasin | 20 U/ µl | 0.2 | 100 µl | |
| Triton X-100 | 10% | 0.1 | 100 µl | |
| NIM2 | 9.7 ml | |||
| Total | 10 ml |
Wash Buffer
| A | B | C | D | |
| Start conc | Final | Volume/amount | ||
| BSA | 1% | 0.1 g | ||
| RNasin | 40 U/ µl | 0.2 | 50 µl | |
| 1xPBS | 10% | 9.9496 ml | ||
| Total | 10 ml |
Diluted Nuclei Buffer
Prepare this buffer while centrifuging nuclei in step 11
| A | B | |
| 20x Nuclei buffer | 25 µl | |
| DTT | 0.5 µl | |
| RNase Inh | 12.5 µl | |
| Nuclease free water | 460 µl |
90% Percoll solution
Prepare fresh and keep on ice
| A | B | |
| Percoll | 4.5 mL | |
| 10x PBS | 0.5 mL |
Clean Dounce homogenizers and pestle B with 70 % ethanol, then Milli-Q water. Place on ice with 50 ml Falcon tube to pre-chill
Collect tissue on dry ice from -80 °C freezer and proceed immediately
Transfer tissue biopsy to homogenizer using sterile, disposable tweezers. If the piece of tissue is in OCT, cutted slides should be directly transferred to the dounce homogenizer already seating on dry ice
Mix Homogenization buffer and transfer 3 mL to the homogenizer making sure to wash the tissue to the bottom of the tube. Wait until buffer is liquid again
Gently homogenize the tissue using the tight pestle (B). Up and down but do not grind. Try to avoid bubbles. 10-20 strokes depending on how the tissue behaves. Rinse the pestle with 0.5 mL of Homogenization buffer. Place the pestle in Virkon for cleaning.
Pour homogenate through a 40 µm cell strainer into 50 ml Falcon tube on ice. Rinse homogenizer with additional 0.5 mL Homogenization buffer and pour it through the strainer. Make sure to transfer droplets of the filtrate from the under side of the strainer using a pipette.
Glass homogenizers should be left for 24 hours in Virkon, then cleaned with 70 % ethanol and Milli-Q water.
Centrifuge the 50 ml Falcon tube @ 500 g, 6 mins, 4 °C with acceleration set at 0 and deacceleration set to 3 in Eppendorf centrifuge.
Carefully remove most of the supernatant with 1 ml pipette tip. Leave a small amount and gently resuspend the nuclei using a wide bore tip (optional). Add 0.5 mL of Wash buffer and carefully resuspend nuclei and transfer to an 1.5 ml eppendorf (NOT low bind).
Check how is the nuclei quality using a C-Chip (5 µl filtered Trypan blue, 5 µl nuclei suspension)
If the nuclei solution is clean enough and not many debris present, add 0.5 mL more of Wash buffer and go to step 12
If many debris is present, start Percoll clean-up:
- Prepare 90% Percoll solution
- Prepare 2 epp with 475 µL of Wash Buffer
- Add 225 µL of the nuclei solution to each one of the epp
- Add 300 µL of 90% Percoll solution to each one of the epp
- Mix by inverting eppendorfs 5 times
- Centrifuge @ 20,000 g, 15 min, 4 °C
- Take supernatant without touching pellet that would be "floating" in the middle-lower part of the tube
- Add 500-900 µl Wash Buffer
- Centrifuge @ 500 g, 3 min, 4 °C
- Take as much supernatant as possible and add 500 µl Wash Buffer in each epp and combine them in one
- Centrifuge @ 500 g, 3 min, 4 °C
-Take most of the supernatant leaving 50-100 µl for resuspension.
- Count nuclei using a C-Chip (5 µl filtered Trypan blue, 5 µl nuclei suspension)
- Concentrate down if necessary, centrifuging at 500 g, 3 mins, 4 °C and taking out extra supernatant volume according to desirable nuclei stock concentration versus targeted nuclei recovery.
- Exemple if the nuclei stock is in100 µl:
add 5 µl 20x Nuclei buffer
add 1 µl DTT(diluted 1:10 in water)
add 2.5 µl RNase Inhibitor????
Centrifuge at 500 g, 3 mins, 4 °C. During centrifuge time, prepare Diluted Nuclei Buffer
Remove all supernatant and resuspend nuclei in the remaining (~100 µl) wash buffer. Check how is the nuclei quality using a C-Chip (5 µl filtered Trypan blue, 5 µl nuclei suspension)
Skip filtering with Flowmi filter if there is not a lot of debris.
Adjust here depending on the size and appearance of the nuclei. If there is enough nuclei density, resuspend in 100 µL of Diluted Nuclei Buffer
Check the nuclei in the new buffer using a C-Chip (5 µl filtered Trypan blue, 5 µl nuclei suspension). Normally will appear clumpy in this buffer!!!!
Filter with a 40 µm Flowmi cell strainer and check nuclei suspension again (a little bit less volume due to filtering)
Count nuclei using a C-Chip (5 µl filtered Trypan blue, 5 µl nuclei suspension). Concentrate down if necessary, centrifuging at 500 g, 3 mins, 4 °C and taking out extra supernatant volume according to desirable nuclei stock concentration versus targeted nuclei recovery.
Follow 10X Single Cell Multiome ATAC + Gene Expression kit protocol (isothermal incubation + GEM generation and barcoding + GEM incubation + quenching reaction)
Keep GEMs at -80 °C