Feb 19, 2025
Nuclei Isolation Protocol Multiome 10X Genomics
- 1University of California San Diego
- Telese Lab
Protocol Citation: Narayan Pokhrel, Francesca Telese 2025. Nuclei Isolation Protocol Multiome 10X Genomics. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbzd41gpk/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 20, 2024
Last Modified: February 19, 2025
Protocol Integer ID: 97009
Keywords: Nuclei isolation, Frozen brain tissues, 10 X Genomics Multiome Analysis
Funders Acknowledgements:
Single-cell resolution analysis of chromatin accessibility and gene expression changes in a model of drug addiction
Grant ID: U01DA050239
Abstract
Nuclei were isolated from frozen rat brain tissue for Chromium Next GEM Single Cell Multiome ATAC + Gene Expression assay (10 X Genomics). The protocol allowed us to obtain good quality individual nuclei with intact nuclei membrane. The isolated nuclei were assessed for transposition reaction followed by GEM (Gel beads-in-emulsion) generation and barcoding in order to simultaneously profile epigenetic landscape and gene expression in the same nuclei. The protocol is optimized to prevent wetting failure due to nuclei clogging during GEM generation step and to reduce detection of ambient RNA.
Guidelines
Procedural guidelines
- Use RNaseZap‱ RNase Decontamination Solution (Cat. no. AM9786) to remove RNase contamination of working station and non-disposable items such as centrifuges and pipettes used during nuclei isolation.
- Perform all steps on ice if possible unless otherwise noted.
- Use disposable, RNase-free pipettes tips, and tubes. If possible, use broad tips as per the need to prevent nuclei damage during pipetting.
- High quality nuclei with well-preserved round morphology are optimal for single-cell multiome library preparation.
Materials
- HB Buffer with IGEPAL
- ATAC-RSB Buffer (without IGEPAL)
- Sucrose
- PBS containing 1% BSA
- ATAC permeabilization buffer (IGEPAL + Digitonin)
- Sterile water
- 70% Isopropyl alcohol
- Dounce Tissue Grinder Set
- 70 um Flomi cell strainers
- Eppendorf LoBind tubes (2 mL and 1.5 mL)
- Swinging-bucket centrifuge
- Pipettes and tips
- Ice
Safety warnings
Perform all steps on ice or at 4 ºC to preserve cell integrity.
- Perform all steps on ice or at 4 ºC to preserve cell integrity.
- Nuclei sticking together could be a sign of overlysis. Thus optimize the protocol before proceeding to GEM (Gel beads-in-emulsion) generation steps.
Ethics statement
Procedures using animals were approved by our Institutional Animal Care and Use Committee (IACUC)
Before start
Preparation: Maintain Cold Conditions: Perform all steps on ice or at 4 ºC to preserve cell integrity.
Procedures of nuclei isolation from brain tissues for 10 X Genomics Chromium Next GEM Single Cell Multiome ATAC + Gene Expression assay
Procedures of nuclei isolation from brain tissues for 10 X Genomics Chromium Next GEM Single Cell Multiome ATAC + Gene Expression assay
Sample Preparation: Maintain Cold Conditions: Perform all steps on ice or at 4ºC to preserve cell integrity
Homogenization:
5m
Use 450 µL of cold Homogenization buffer (HB buffer) with IGEPAL for each sample. Homogenize the tissue using a 2ml Dounce homogenizer with pestle "A" for 10-12 strokes followed by pestle "B" for 10-12 strokes, ensuring to push down and twist.
1m
Incubation and Filteration:
5m 30s
Incubate the homogenate for 2 minutes on ice. Gently mix by pipetting up and down, repeating this process two more times during the incubation period.
2m
Filter the homogenate using a 70 µm Flowmi cell strainer.
2m
Collect the filtered suspension into a pre-chilled 2 mL LoBind tube.
30s
Add 550 µL of HB buffer (without IGEPAL) and mix well before centrifugation.
30s
Centrifugation:
5m
Centrifuge at 500 RCF for 5 minutes at 4ºC to pellet nuclei. Carefully remove the supernatant, leaving behind approximately 50 µL if the pellet is not clearly visible. Keep the supernatant on ice for later steps, if quality check for nuclei loss is needed.
Sucrose Cushion
35m
Resuspend the nuclei in 1 mL of ATAC-RSB buffer (without IGEPAL).
Carefully underlay with 400 µL of ATAC-RSB buffer containing 1.5 M Sucrose.
Look for the separation of layers and then centrifuge at 5500Xg for 30 minutes at 4ºC.
Nuclei permeabilization
20m
Remove most of the supernatant, being careful not to disturb the pellet. Leave behind 50-200 µLif necessary.
Resuspend the nuclei in 450 µL of ATAC permeabilization buffer and incubate for 1 minute on ice.
Filter the suspension using a 40 µm Flowmi cell strainer into a new 1.5 mL LoBind Eppendorf tube.
Add 550 µL of PBS containing 1% BSA, centrifuge at 500 RCF for 5 minutes at 4ºC, discard the supernatant, and resuspend the nuclei in 300 µL of the same PBS solution + 1%BSA.
After centrifugation, remove the supernatant, leaving 10-20 µL for resuspension with 200 µL of 1X Diluted Nuclei Buffer (PN-2000207, 10X Genomics Kit).
Centrifuge at 350 RCF for 5 minutes at 4ºC.
After a final centrifugation, remove the supernatant, leaving 5-6 µL for resuspension.
Nuclei Counting:
Prepare Hoechst Solution: Mix 1 µL of Hoechst stock solution (1 mg/mL in PBS) with 99 uL of PBS.
30m
Counting: Prepare a dilution with 0.5 µL of the resuspended nuclei, 1 µL PBS + Hoechst and 8.5 µL of PBS. Place 10 µL on a counting chamber and count under a microscope, covering nine fields to calculate average and standard deviation. Take images to assess the nuclei status and to keep record.
Evaluating the quality of nuclei for single-cell multiome analysis: Nuclei with intact round morphology are regarded as high quality and are optimal for single-cell multiome library preparation. A. Brighfield image of brain nuclei, B. DAPI staining of brain nuclei, C. Merged image of brightfield and DAPI channel.
Calculation: Use the provided formula to determine the total number of nuclei. Total nuclei = (Average nuclei count in fields)* (final resuspension volume of nuclei * 10). 12,000-14,000 nuclei are loaded during GEM generation step in order to obtain a target of 6,000 nuclei for further analysis.
Stock Buffers and Solutions:
Stock Buffers and Solutions:
30m
30m
Preparation: All stock solutions should be filtered using a 0.22 µm PVDF filter system. Stock solutions are stable at 4ºC for at least 6 months.
| A | B | C | D | E | |
| 1.034x Homogenization Buffer Stable Solution | For 200 ml stock solution | ||||
| Stock | Name | Final Conc. | Fold Dilution (x) | Total Vol. (μl) | |
| 1 M | Sucrose | 0.26 M | 3.87 | 51706.50 | |
| 2 M | KCl | 0.03 M | 77.36 | 2585.33 | |
| 1 M | MgCl2 | 0.01 M | 193.40 | 1034.13 | |
| 0.75 M | Tricine-KOH pH 7.8 | 0.02 M | 36.26 | 5515.36 | |
| - | Water | - | - | 139158.69 | |
| Total Volume (µL) | 200000.00 | ||||
| A | B | C | D | E | |
| Stable ATAC-RSB Buffer | For 500 mL stock solution | ||||
| Stock | Name | Final Conc. | Fold Dilution (x) | Total Vol. (μl) | |
| 1 M | Tris-HCl pH 7.5 | 0.01 M | 100.00 | 5000.00 | |
| 5 M | NaCl | 0.01 M | 500.00 | 1000.00 | |
| 1 M | MgCl2 | 0.003 M | 333.33 | 1500.00 | |
| - | Water | - | - | 492500.00 | |
| Total Vol. (µL) | 500000.00 |
| A | B | C | D | E | F | |
| HB unstable buffer (without IGEPAL) [Prepare 2.2 mL per 1-2 sample (500 µLw/o IGEPAL +500 µL with IGEPAL 10% extra] | ||||||
| Reagents | Stock conc. | Final conc | 1-2X | 1-2X (10% extra) | 4X | |
| HB stable solution | 1.0341X | 1 | 1954 | 2149.4 | 4298.8 | |
| DTT | 1 M | 1 mM | 2 | 2.2 | 4.4 | |
| Spermidine | 0.5 M | 0.5 mM | 2 | 2.2 | 4.4 | |
| Spermine | 0.15 M | 0.15 mM | 2 | 2.2 | 4.4 | |
| Pierce Proteinase Inhibitor | 50X | 1X | 40 | 44 | 88 | |
| Total | 2000 µL | 2200 µL | 4000 µL |
Note: Add RNase protector (40 U/ µL) at the last step. 12.5 µL per mL of buffer.
| A | B | C | D | E | F | G | |
| HB unstable buffer (with IGEPAL) (calculation is done for 10% extra reagent/sample) | |||||||
| Reagents | Stock conc. | Final conc | 1X | 2X | 3X | 4X | |
| HB unstable solution (w/o IGEPAL) | 1.0341X | 1 | 544.5 µL | 1089 | 1633.5 | 2178 | |
| IGEPAL | 10% | 0.1% | 5.5 µL | 11 | 16.5 | 22 | |
| Total | 550 µL | 1100 | 1650 | 2200 |
Note: Add RNase protector (40 U/ µL) at the last step. 12.5 µL per ml of buffer.
| A | B | C | D | E | F | G | |
| Preparation of 1.5 M sucrose in ATAC RSB buffer (without IGEPAL): For 4 samples, prepare in total volume of 2 mL | |||||||
| Reagents | Stock conc. | Final conc | 1X | 2X | 3X | 4X | |
| ATAC RSB buffer (w/o IGEPAL) | 1.0341X | 1 | 500 µL | 1000 µL | 1500 µL | 2000 µL | |
| Sucrose | 1.5 M | 0.256 gm | 0.513 gm | 0.768 gm | 1.0269 | ||
| Total | 500 µL | 1000 µL | 1500 µL | 2000 µL |
Note:
1 . Vortex for 2 min in order to dissolve sucrose in ATAC RSB buffer. After no crystals are visible, place the sucrose solution in ice.
2. Add RNase protector (40 U/ µL) at the last step. 12.5 µL per ml of sucrose solution.
| A | B | C | D | |
| ATAC permeabilization buffer | ||||
| Stock Conc. | Final conc. | For 4 samples | ||
| ATAC-RSB | 2994 µL | |||
| IGEPAL | 10% | 0.01% | 2 µL | |
| Tween | 10% | 0.01% | 2 µL | |
| Digitonin | 5% | 0.001% | 0.4 µL | |
| DTT | 1000 mM | 1 mM | 2 µL | |
| TOTAL | 2000 µL |
Note: 1. Add RNase protector (40 U/ µL) at the last step. 12.5 µL per ml of buffer.
| A | B | C | D | |
| 1X Diluted Nuclei Buffer | ||||
| Diluted Nuclei Buffer: Maintain at 4 degree Celcius | Stock | Final | 1 mL | |
| 20X Nuclei Buffer (PN-2000207) | 20X | 1X | 50 uL | |
| DTT | 1,000 mM | 1 mM | 1 uL | |
| RNase Inhibitor (Confirm vendor-specific stock concentration) | 40 U/ µL | 1 U/ µL | 25 µL | |
| Nuclease-free Water | - | - | 924 µL |
Reagent Order Lists
Reagent Order Lists
Order List
| A | B | C | |
| Item | Supplier | Cat Number | |
| Eppendorf 2 mL Lo-Bind tubes | Sigma | Z666556-250EA | |
| Eppendorf 1.5 mL Lo-Bind tubes | Sigma | Z666548-250EA | |
| Nunc cryovials | Thermo | 375418PK | |
| Iodixanol (comes at 60%) | Sigma | D1556-250ML | |
| Sucrose | Sigma | S7903-250G | |
| NP40 | Roche (Sigma) | 11332473001 | |
| Tricine | Sigma | T0377-25G | |
| Potassium Hydroxide (KOH) | Sigma | P5958-250G | |
| Complete Protease Inhibitors | Roche | 11697498001 | |
| MgCl2 | Ambion (Thermo) | AM9530G | |
| KCl | Ambion (Thermo) | AM9640G | |
| DTT | Thermo | R0861 | |
| Spermidine | Sigma | S2501 | |
| Spermine | Sigma | S3256-1G | |
| 70 µm Flowmi cell strainers | Fisher | 03-421-228 | |
| 70 µm bucket-style cell strainers | BD Falcon | 352350 | |
| Tris-HCl pH 7.5 | Invitrogen | 15567-027 | |
| NaCl | Ambion (Thermo) | AM9759 | |
| Tween 20 | Roche (Sigma) | 11332465001 | |
| H2O | Invitrogen | 10977-015 | |
| Dounce Tissue Grinder Set | Sigma | D8938-1SET | |
| INCYTO Disposable hemocytometers | Fisher | 22-600-100 | |
| BAM Banker | Wako Chemicals | 302-14681 | |
| RiboLock | Thermo | EO0384 | |
| 0.22 µm PVDF Filter Units (500 ml) | Millipore | SCGVU05RE | |
| 0.22 µm PVDF Filter Units (50 ml) | Millipore | SE1M179M6 | |
| RNase inhibitor | Roche | 03335402001 | |
| RNaseZap TM RNase Decontamination solution | Invitrogen | AM9786 |