Dec 04, 2025
Single-Nuclei Isolation from Adult Mouse Ventral Midbrain
- Yue Ma1
- 1Van Andel Institute
Protocol Citation: Yue Ma 2025. Single-Nuclei Isolation from Adult Mouse Ventral Midbrain. protocols.io https://dx.doi.org/10.17504/protocols.io.14egnr8kml5d/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: July 19, 2025
Last Modified: December 04, 2025
Protocol Integer ID: 222827
Keywords: ASAPCRN, nuclei isolation from adult mouse ventral midbrain, single nuclei from frozen tissue, adult mouse ventral midbrain, chromium fixed rna kit, compatible with chromium fixed rna kit, nuclei isolation, single nuclei, 10x genomic, frozen tissue
Funders Acknowledgements:
ASAP
Grant ID: ASAP-000592
Abstract
We use this protocol to isolate single nuclei from frozen tissue. It is compatible with Chromium Fixed RNA Kit (10x Genomic).
Materials
Required buffers and reagents:
1. Homogenize buffer I
- HBSS (Thermo Fisher 14175095)
- 0.2 U/ul RNase Inhibitor (40U/ul, Millipore Sigma, 3335402001)
2. Nuclei Extraction Buffer (Miltenyi Biotec, 130-128-024)
3. Homogenize buffer II (HMII)
- 0.25 M sucrose
- 25 mM KCL
- 5 mM MgCl2
- 20 mM Tris-HCL
- pH7.8
4. 50% Iodixanol working solution (WS)
- 5 vol of OptiPrep
- 1 vol of the diluent (150mM KCL, 30mM MgCl2, 120 mM Tris-HCL, pH7.8)
5. 40% Iodixanol-Sucrose-KCL-MgCL2 solutions
- 4 vol of WS (800ul/sample)
- 1 vol of HMII (200ul/sample)
6. Nuclei Wash and Resuspension Buffer
- 1X PBS (Life Technologies: 10010-023)
- 1% BSA (ThermoFisher: AM2616)
- 0.2 U/ul RNase Inhibitor (Millipore Sigma, 3335402001)
- Ultrapure Water (Life Technologies: 10977-015)
7. Fixation Buffer
- 4% Formaldehyde
- Conc. Fix & perm buffer (10x genomics PN 2000517)
- Ultrapure Water
8. Quenching Buffer
- Conc. Quench Buffer (10x Genomics PN 2000516)
- Ultrapure Water
9. Enhancer
10. 50% glycerol
Others:
1. Centrifuge with fixed angle rotor (3000rcf, 4 °C)
2. Centrifuge with swing bucket rotor (1.5ml/15ml adaptor, 500rcf, 4 °C)
3. Dry ice for tissue short storage
4. Wet ice for whole process
5. 15ml tubes/ 2-ml sharp bottomed tubes/ 1.5-ml low binding tubes
6. Hand-held TissueRuptor (TEM: 22573)
7. Disposable tips (STERILE OMNI TIP)
8. Wide-bore glass pipette (pre-autoclaved)
9. 40 µm-strainer (with adapter)
10. Trypan blue
11. Cell counter
12. 200ul/1000ul pipette & tips with filter.
13. Heat bath (65°C)
14. 70% ethanol
15. RNase ZIP
16. PPE (gloves/masks)
17. Trash beaker
Troubleshooting
Before start
(Always keep in mind of RNase free)
Dissociation & Release Nuclei
Add 3ml Homogenize buffer I to 15ml tube, transfer tissue into the tube, homogenize on ice with hand-held TissueRuptor (TEM: 22573) with disposable tips (STERILE OMNI TIP), at lowest setting, 5 sec on, 5 sec off, repeat 4 times, store on ice.
Centrifuge the homogenates at 500rcf for 5 minutes at 4°C and remove supernatant.
Add 3 mL chilled Nuclei Extraction Buffer (Miltenyi Biotec, 130-128-024) to the tissue. Resuspend the pellet using a wide-bore glass pipette, mix gently and incubate on ice for 15min. Triturate 10x by pipetting up and down with wide-bore glass pipette during the incubation.
Filter homogenate using a 40 µm-strainer (with adapter) to a new 15ml tube. (Optional: check residue on the strainer, consider recover it). Centrifuge the nucleus at 500rcf for 5 minutes at 4°C and remove supernatant.
Add 900 uL homogenate buffer II, resuspend the pellet and mix 10x using a regular-bore pipette tip.
Proceed to Nucleus Purification.
Nucleus Purification (Optiprep, 60% Iodixanol)
Add 900uL 40% Iodixanol to 900uL homogenates, mix well and transfer the sample into 2-ml sharp bottomed tube.
Centrifuge the mixture containing the nucleus at 3000 rcf for 30 min at 4°C.
Carefully (!!!) remove supernatant (watch out for cell debris on the tube wall), using a regular-bore pipette tip, add 200uL Nuclei Wash and Resuspension Buffer, resuspend the nuclei pellets, transfer to a new 1.5-ml low binding tube.
Add 800uL Nuclei Wash and Resuspension Buffer. Gently suspend the pellet.
Check the quality and purity of the nucleus (Target nuclei concentration is 700-1,200 nuclei/µL). QC can be done with microscope/ trypan blue cell counting/ fluorescence cell counting (Luna cell counter).
Centrifuge nuclei at 500 rcf for 5min at 4°C (Swinging bucket !!!).
(optional) repeat washing step 4) for 1-2 times in total.
Proceed to fixation.
Nuclei Fixation/ Storage
Add 1 ml Fixation Buffer to the sample pellet and pipette mix 5x.
Incubate for 16-24 h at 4°C.
*DO NOT agitate or mix the sample during incubation.
*Fixation time and temperature should be consistent across all samples in an experiment.
Centrifuge at 500 rcf for 5 min at room temperature (Swinging bucket !!!). Remove the supernatant without disturbing the pellet.
Add 1 ml chilled Quenching Buffer to the sample pellet and pipette mix 5x and keep on ice.
Thaw Enhancer (included in the 10x Genomics Kit_PN-1000414) for 10 min at 65°C. Vortex and centrifuge briefly. Keep warm and verify no precipitate before use. (Once thawed, Enhancer can be kept at 42°C for up to 10 min.)
Add 0.1 volume pre-warmed Enhancer to fixed nuclei in Quenching Buffer (included in the 10x Genomics Kit PN-1000414).
Add 50% glycerol for a final concentration of 10%. Pipette mix. Store at -80°C for up to 6 months.