Nov 21, 2025
Midbrain sample preparation for 10xGenomics Multiome Protocol
- Matthew Kearney1,2,
- Zhixiang Liao1,3,
- Idil Tuncali1,3,
- Nathan Haywood1,4,
- Monika Sharma1,2,
- Madison Cline1,5,
- Geidy E Serrano1,5,
- Thomas G Beach1,5,
- Joshua Z Levin1,4,
- Clemens Scherzer1,2,6,7,8
- 1Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD 20815, USA;
- 2Stephen & Denise Adams Center for Parkinson’s Disease Research of Yale School of Medicine, New Haven, CT 06510;
- 3Brigham and Women's Hospital;
- 4Stanley Center for Psychiatric Research, Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA;
- 5Banner Sun Health Research, Sun City, AZ 85351, USA;
- 6APDA Center for Parkinson Precision Medicine, Yale, New Haven, CT 06510;
- 7Department of Neurology, Yale, New Haven, CT 06510;
- 8Department of Genetics, Yale, New Haven, CT 06510
- Scherzer Lab
Protocol Citation: Matthew Kearney, Zhixiang Liao, Idil Tuncali, Nathan Haywood, Monika Sharma, Madison Cline, Geidy E Serrano, Thomas G Beach, Joshua Z Levin, Clemens Scherzer 2025. Midbrain sample preparation for 10xGenomics Multiome Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.14egnr17zl5d/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: October 29, 2025
Last Modified: November 21, 2025
Protocol Integer ID: 231075
Keywords: midbrain sample preparation for 10xgenomics multiome protocol, 10xgenomics multiome protocol, processing of human frozen postmortem midbrain tissue, human frozen postmortem midbrain tissue, midbrain sample preparation, nuclei multiome library generation, rnaseq, isolating single nuclei, single nuclei, nuclei
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000301
Abstract
This protocol describes the processing of human frozen postmortem midbrain tissue for isolating single nuclei. The nuclei are purified and sorted and subjected to 10X single-nuclei multiome library generation and sequencing (RNAseq + ATAC-seq). Four samples can be processed simultaneously with this protocol.
Materials
1. 20X Nuclei Buffer (10x Genomics, 2000207, Supplied in Multiome Kit), -20°C
2. Sigma Protector RNase Inhibitor (Sigma,3335402001), -20°C
3. Digitonin (Thermo Fisher, BN2006), 4°C, incubate at 65 °C before use
4. 1000mM DTT (Sigma, 43816-10ml), 4°C, freshly dilute to 100mM with water
5. 10% MACS BSA Stock Solution (Miltenyi Biotec, 130-091-376), 4°C
6. 1mg/ml 7-ADD Solution (Invitrogen, A1310)
7. RNase-Free Disposable Pellet Pestles (Fisher Scientific, 12-141-368), RT
8. DPBS (Gibco, 14190-144), RT
9. Sodium Chloride (Fisher, S671), RT
10. Magnesium Chloride (Sigma, M9272), RT
11. 1M pH7.4 Trizma Hydrochloride Solution (Sigma, T2196), RT
12. 100% Nonidet P40 Substitute (Sigma, 74385), RT, dilute to 10% with water
13. 10% Tween 20 (Bio-Rad, 1662404), RT
14. 70µm Nylon Mesh (Fisher, 22363548), RT
15. 40µm Flowmi Cell Strainer (Bel-Art, 136800040), RT
16. Wide bore tips (Thermo Scientific, 2079G), RT
| A | B | C | |
| All Buffer Amounts Sufficient for Processing 4 Samples | |||
| NP40 Lysis Buffer (NPLB) | |||
| Stock | Amount | ||
| pH 7.4 Tris-HCl | 1M | 57µl | |
| NaCl | 1M | 57µl | |
| MgCl2 | 1M | 17.1µl | |
| Nonidet P40 Substitute | 10% (Diluted from 100% with water before use) | 57µl | |
| DTT | 100mM (Diluted from 1M with water before use) | 57µl | |
| RNase Inhibitor | 40U/µl | 142.5µl | |
| Nuclease-free Water | 5.31ml | ||
| Total | 5.7mL | ||
| PBS Buffer (PBSB) | |||
| Stock | Amount | ||
| BSA | 10% | 1ml | |
| RNase Inhibitor | 40U/µl | 250µl | |
| DPBS | 8.75ml | ||
| Total | 10ml | ||
| 1X Lysis Buffer (LB) | |||
| Stock | Amount | ||
| pH 7.4 Tris-HCl | 1M | 10µl | |
| NaCl | 1M | 10µl | |
| MgCl2 | 1M | 3µl | |
| Tween-20 | 10% | 10µl | |
| Nonidet P40 Substitute | 10% (Diluted from 100% with water before use) | 10µl | |
| Digitonin (Incubate at 65°C before use) | 2µl | ||
| BSA | 10% | 100µl | |
| DTT | 100mM (Diluted from 1M with water before use) | 10µl | |
| Nuclease-free Water | 845µl | ||
| Total | 1ml | ||
| Lysis Dilution Buffer (DLB) | |||
| Stock | Amount | ||
| pH 7.4 Tris-HCl | 1M | 10µl | |
| NaCl | 1M | 10µl | |
| MgCl2 | 1M | 3µl | |
| BSA | 10% | 100µl | |
| DTT | 100mM (Diluted from 1M with water before use) | 10µl | |
| Nuclease-free Water | 867µl | ||
| Total | 1ml | ||
| 0.1X Lysis Buffer (0.1XLB) | |||
| Stock | Amount | ||
| LB | 44µl | ||
| Rnase Inhibitor | 40U/µl | 11µl | |
| DLB | 385µl | ||
| Total | 440µl | ||
| Wash Buffer (WB) | |||
| Stock | Amount | ||
| pH 7.4 Tris-HCl | 1M | 22µl | |
| NaCl | 1M | 22µl | |
| MgCl2 | 1M | 6.6µl | |
| BSA | 10% | 220µl | |
| Tween-20 | 10% | 22µl | |
| DTT | 100mM (Diluted from 1M with water before use) | 22µl | |
| Rnase Inhibitor | 40U/µl | 55µl | |
| Nuclease-free Water | 1.83ml | ||
| Total | 2.2ml | ||
| Diluted Nuclei Buffer (DNB) | |||
| Stock | Amount | ||
| 20X Nuclei Buffer | 5µl | ||
| DTT | 100mM (Diluted from 1M with water before use) | 1µl | |
| Rnase Inhibitor | 40U/µl | 2.5µl | |
| Nuclease-free Water | 91.5µl | ||
| Total | 100µl |
Protocol materials
7-AAD (7-Aminoactinomycin D)Thermo FisherCatalog #A1310
Cryosectioning
Frozen Brain Samples were received from the brain bank - Four 50µm sections were collected in a 1.5 ml microcentrifuge tube and shipped on dry ice to the Lab.
Prepare Buffers and Equipment
All buffers are prepared the day of the experiment. DTT and RNAse inhibitor are added to the buffers immediately before using. Prepare & maintain all buffers On ice . See all buffer recipes in the Materials tab.
Pre-cool the microcentrifuge to 4 °C
Nuclei Isolation and Sorting
25m
Add 650 µL NPLB to the tube with tissue and homogenize 15x using a pestle On ice .
Add 650 µL NPLB to the tube and incubate for 00:05:00 On ice , pipette mix a few times during incubation with wide bore tips (use in step 4, 5 and 6).
5m
Pass the suspension through a 70 μm Nylon Mesh into a pre-chilled 50 ml Falcon tube On ice .
Transfer the filtered lysate to a pre-chilled Lo-Bind 2 ml tube On ice .
Centrifuge at500 rcf, 4°C, 00:05:00 .
5m
Remove most of the supernatant, leaving around50 µL .
Add 1 mL PBSB, DO NOT mix. Incubate for 00:05:00 On ice .
Pipette mix to resuspend the pellet
Centrifuge at 500 rcf, 4°C, 00:05:00 .
5m
Remove most of the supernatant, leaving around50 µL .
Resuspend with 1.4 mL PBSB.
Add 1.4 µL 7-AAD (7-Aminoactinomycin D)Thermo FisherCatalog #A1310 to the 1.4 mL sample, mix by inverting.
Incubate for 00:05:00 On ice .
5m
Pass the suspension through a 40 µm FlowMi strainer into a pre-chilled 5 ml FACS tube On ice :
Aspirate 700 µL of the suspension into a regular 1 mL pipette tip. Add the FlowMi filter to the end of the tip. Press the plunger slowly to dispense into the new tube. Discard tip and filter.
Repeat step 17.1 one additional time for the remaining volume.
Pre-load 1.5 ml microcentrifuge tubes with 15 µL PBSB for nuclei collection. Maintain On ice .
Sort nuclei using a 100 µm nozzle on BD FACSAria or similar. Collect sorted nuclei in chilled, pre-loaded 1.5 ml microcentrifuge tubes.
Note
Gate first by SSC and FSC to remove doublets. Then, collect 7-AAD positive events - these are nuclei.
Nuclei Permeabilization
12m
Centrifuge sorted nuclei at 500 rcf, 4°C, 00:05:00
5m
Remove the supernatant without disrupting the nuclei pellet.
Resuspend the pellet in 100 µL 0.1XLB and pipette mix 5x.
Incubate for 00:02:00 On ice .
2m
Add 500 µL WB and pipette mix 5x.
Centrifuge at 500 rcf, 4°C, 00:05:00 .
5m
Remove the supernatant without disrupting the nuclei pellet.
Based on the nuclei count post-sorting & targeted nuclei recovery, resuspend in chilled DNB.
Note
See table in Step 1.1 of the 10X Multiome User Guide (Rev E) for help with calculating DNB volume.
Note
Based on empirical data, actual nuclei counts are approximately 1/3rd of collected FANS events.
Proceed immediately to Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide (CG000338 Rev E, page 30). Library preparation and sequencing for both RNAseq + ATAC-seq is performed follwing strictly 10x Genomics protocol (CG000338 Rev E).