Nuclear extraction using opti-prep gradient centrifugation for 10X Genomics Multiome Assay

Lite Yang; Pauline Meriau; Valeria Cavalli; Robert W. Gereau

Jul 21, 2025

Nuclear extraction using opti-prep gradient centrifugation for 10X Genomics Multiome Assay

DOI

https://dx.doi.org/10.17504/protocols.io.5jyl8q4y8l2w/v1

Lite Yang¹,
Pauline Meriau²,
Valeria Cavalli^2,3,4,
Robert W. Gereau^1,2

¹Department of Anesthesiology, Washington University Pain Center, Washington University School of Medicine, St. Louis, MO, United States;
²Department of Neuroscience, Washington University School of Medicine, St. Louis, MO, United States;
³Center of Regenerative Medicine, Washington University School of Medicine, St. Louis, MO, United States;
⁴Hope Center for Neurological Disorders, Washington University School of Medicine, St. Louis, MO, United States

Pauline Meriau

Department of Neuroscience, Washington University School of ...

DOI: https://dx.doi.org/10.17504/protocols.io.5jyl8q4y8l2w/v1

Protocol Citation: Lite Yang, Pauline Meriau, Valeria Cavalli, Robert W. Gereau 2025. Nuclear extraction using opti-prep gradient centrifugation for 10X Genomics Multiome Assay. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8q4y8l2w/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: November 11, 2024

Last Modified: July 21, 2025

Protocol Integer ID: 220552

Keywords: Hemocytometer, Epifluorescence microscope, Homogenization, prep gradient centrifugation for 10x genomics multiome assay, 10x genomics multiome assay, nuclear extraction, gradient centrifugation, using opti

Abstract

This protocol describes the nuclear extraction by using opti-prep gradient centrifugation for 10X Genomics Multiome Assay.

Materials

Equipment and Materials:

Bench-top centrifuge
Swing-bucket rotor ultracentrifuge (Beckman, L-70)
Swing-bucket rotor (Beckman, SW 28)
Ultracentrifuge tube (4 ml thick-walled polycarbonate tube, Beckman Coulter 355645)
Equipment
4 mL, Open-Top Thickwall Polycarbonate Tube
NAME
Centrifuge tube
TYPE
Beckman Coulter
BRAND
355645
SKU
https://www.mybeckman.in/supplies/tubes-and-bottles/open-top-tubes/355645
LINK

Ultracentrifuge rotor adaptor (13mm Diameter Delrin Tube Adapter, Beckman Coulter 303392)
Equipment
13mm Diameter Delrin Tube Adapter
NAME
Ultracentrifuge Rotors
TYPE
Beckman Coulter
BRAND
303392
SKU
https://www.mybeckman.in/supplies/adapters/303392
LINK

2 ml Dounce Tissue Grinder (Sigma-Aldrich, D8938-1SET)
Equipment
Dounce tissue grinder set
NAME
Tissue grinder
TYPE
KIMBLE 
BRAND
D8938
SKU
https://www.sigmaaldrich.com/IN/en/product/sigma/d8938?srsltid=AfmBOor_tbRsOVPIEmWp6R9nh7fQDqXh4WQFaYkkhyTHiIWGNG1n7RVw
LINK

5 ml DNA LoBind tube (Eppendorf, 0030108310)
Equipment
DNA LoBind® Tubes, 5 mL
NAME
Eppendorf Tubes
TYPE
Eppendorf
BRAND
0030108310
SKU
https://www.eppendorf.com/in-en/Products/Laboratory-Consumables/Tubes/DNA-LoBind-Tubes-p-0030108310
LINK

2 ml DNA LoBind tube (Eppendorf, 022431048)
1.5 ml DNA LoBind tube (Eppendorf, 022431021)
5 mL Round Bottom High Clarity PP Test Tube
Equipment
5 mL Round Bottom High Clarity PP Test Tube
NAME
Tube
TYPE
Falcon®  
BRAND
352063
SKU
https://ecatalog.corning.com/life-sciences/b2b/DE/en/General-Labware/Tubes/Tubes,-Round-Bottom/Falcon%C2%AE-Round-Bottom-High-clarity-Polypropylene-Tube/p/352063'' 
LINK

CellTrics® filters (50 um, Sysmex, 04-004-2327)
Pipettes and pipette tips
Hemocytometer


Reagents:

ReagentSucroseFisher ScientificCatalog #AAJ6514836 
ReagentPBS, 10X SolutionFisher ScientificCatalog #AAJ75889AE 
ReagentUltrapure WaterThermofisherCatalog #10977023 
ReagentTris-HCl (ph7.4)Merck MilliporeSigma (Sigma-Aldrich)Catalog #T2194-100ML 
ReagentTris (1 M) pH = 8 RNase freeInvitrogen - Thermo FisherCatalog #AM9855G 
Reagent5M NaClInvitrogen - Thermo FisherCatalog #AM9760G 
Reagent2M KClInvitrogen - Thermo FisherCatalog #AM9640G 
Reagent1M MgCl2Invitrogen - Thermo FisherCatalog #AM9530G 
Reagent10% Tween 20Bio-Rad LaboratoriesCatalog #1662404 
ReagentSurfact-Amps NP-40Thermo Fisher ScientificCatalog #28324 
ReagentDigitonin (5%)Thermo FisherCatalog #BN2006 
Reagent10% Triton X-100Merck MilliporeSigma (Sigma-Aldrich)Catalog #93443-100ML 
ReagentOptiprep ( Iodixanol)Merck MilliporeSigma (Sigma-Aldrich)Catalog #D1556-250ML 
ReagentBovine Serum AlbuminMerck MilliporeSigma (Sigma-Aldrich)Catalog #126625 
ReagentProtector RNase InhibitorMerck MilliporeSigma (Sigma-Aldrich)Catalog #3335402001 
ReagentDAPI Solution (1 mg/mL)Thermo Fisher ScientificCatalog #62248 
Reagent7-ADD Ready Made SolutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #SML1633-1ML 
Reagent1M DL-Dithiothreitol solution (DTT)Merck MilliporeSigma (Sigma-Aldrich)Catalog #646563 
ReagentNuclei Buffer 20X 10x GenomicsCatalog #2000153/2000207 
 

ABCD
PBSStockFinal50 mL
Nuclease-free Water45 mL
PBS10X1X5 mL
  
ABCD
NIM (Nuclear isolation media)StockFinal100 mL
Nuclease-free Water47.25 mL
Sucrose0.5 M0.25 M50 mL
KCl2 M25 mM1250 µL
MgCl21 M5 mM500 µL
Tris-HCl, pH 8.01 M10 mM1000 µL
  
ABCD
DiluentStockFinal50 mL
Nuclease-free Water41.75 mL
KCl2 M150 mM3.75 mL
MgCl21 M30 mM1.5 mL
Tris-HCl, pH 8.01 M60 mM3 mL
  
ABCD
Homogenization BufferStockFinal1100µL per sample
NIM1060 µL
BSA30%1%37 µL
RNase inhibitor40 U/ µL0.08 U/ µL2.2 µL
  
ABCD
Resuspension BufferStockFinal1100µL per sample
PBS1X1X1060 µL
BSA30%1%37 µL
RNase inhibitor40 U/ µL0.08 U/ µL2.2 µL
  
ABCD
Nuclei Permeabilization BufferStockFinal1 ml per 8 samples
Nuclease-free Water963 µL
Tris-HCl (pH 7.4)1M10 mM10 µL
NaCl5M10 mM2 µL
MgCl21M3 mM3 µL
Tween-2010%0.1%10 µL
NP4010%0.1%10 µL
Digitonin5%0.01%2 µL
  
ABCD
Diluted Nuclei BufferStockFinal55µL per sample
Nuclease-free Water--520 µL
Nuclei Buffer (20X)20X1X27.5 µL
DTT1M1mM0.55 µL
RNase inhibitor40U/ µL0.08 U/ µL1.1 µL
 
Opti-prep gradients per sample:
 
AB
50% iodixanol3 mL per sample
Opti-prep2500 µL
Diluent500 µL
  
AB
40% iodixanol1100 µL per sample
50% iodixanol880 µL
NIM220 µL
30% BSA37 µL
RNase inhibitor2.2 µL
  
AB
30% iodixanol1100 µL per sample
50% iodixanol660 µL
NIM440 µL
30% BSA37 µL
RNase inhibitor2.2 µL
 

Troubleshooting

Before start

One day prior to the experiment:

Wipe clean the working area with 70% ethanol and RNase Zap.
PBS, NIM, and Diluent can be prepared in advance and stored at Temperature4 °C   for up to 3 months. Filter NIM with 0.2 µm filter.
Clean douncers, scissors, and forceps with 70% ethanol, and rinse them with nuclease-free water for a total of 2 times.

On the day of the experiment

Gather equipment, materials, and reagents. 

Thaw and dissolve DTT by incubating the tube at TemperatureRoom temperature  .

Place SW28 rotor into the ultracentrifuge, start test run at Centrifigation8900 rpm, 4°C, 01:00:00 .

Pre-chill the bench-top centrifuge to Temperature4 °C  .

Place douncers TemperatureOn ice  .

Prepare buffers and store them TemperatureOn ice  .

Gather samples and store on dry ice. 

Layer in ultracentrifuge tube 1 ml 40% at bottom of tube, and then slowly add on top of it, Amount1 mL   30% iodixanol solutions carefully. Slowly pipette just above 40% layer against the wall allowing it to form two distinct layers. Leave this TemperatureOn ice   while you prepare the sample. Pre-indent ice with another tube to avoid disrupting layers. 

Sample processing

1m 5s

Take one sample out from dry ice and thaw TemperatureOn ice   for Duration00:01:00  .

Add Amount1 mL   of 1X PBS to the original tube, pipet mix 10 times and vortex for Duration00:00:05   to wash off OCT. 

Quick spin and aspire out PBS.

Repeat Steps 6 and 7 one more time.

Using the forceps to transfer the tissue to a 5 mL centrifuge tube.

Add Amount1 mL   of homogenization buffer and Amount10 µL   of 10% NP40.

Break down tissues by chopping the sample 30 times with a clean scissor until all sections have been trimmed into small pieces.

Homogenization

Transfer samples to a pre-chilled douncer.

Insert the loose pestle (“A”) into the homogenizer and perform 15 strokes. Perform additional 15 strokes with the tight pestle (“B”). Try to make less bubbles as possible. 

Note
Optional: if big chunks are still visible, remove the pestle and let the douncer sit TemperatureOn ice   for Duration00:01:00   allowing chunks to precipitate. Perform additional 5 strokes with the tight pestle (“B”). At the end of each stroke, rotate the pestle back and forth to further grind the sample.

Filter the homogenate directly with a 50 µm Sysmex filter into a 2 mL centrifuge tube.

Note
If there is abundant debris, filtration can be aided by taking your thumb and covering the top of the filter opening. This will create positive pressure that pushes the filtrate through the filter. 

Bring the sample volume up to Amount1 mL   using resuspension buffer.

Vortex 50% iodixanol. Add Amount1 mL   of freshly-vortexed 50% iodixanol to the homogenate to make 25% iodixanol. 

Cap the tube and keep TemperatureOn ice  . Repeat all sample preparation steps and Homogenization steps for additional samples.

Centrifugation

28m

Invert sample tubes 10 times to mix up nuclear suspensions in 25% iodixanol.

Using a 1 ml pipette, slowly pipette Amount2 mL   of nuclear resuspensions on top of the 30% layer in the ultracentrifuge tube. Slowly pipette just above 30% layer against the wall allowing it to form two distinct layers.

Adjust volume of samples with nuclease-free water to match same level. 

Insert the ultracentrifuge tube into the adaptor. Seal the top with parafilm.

Spin: Centrifigation10000 x g, 00:18:00  (8,900 rpm with SW28 rotor).

18m

When spin is finished, aspirate out Amount1.5 mL   of 25% iodixanol layer in order to get better access to the interlayer.

Stab P200 pipette the interface of 30% and 40%. While moving pipette tip over the entire surface of this interface, aspirate Amount200 µL   of interlayer and transfer to a 2 mL centrifuge tube. 

Note
Dots of nuclei may be visible interface between 30% iodixanol and 40% iodixanol layers.

Repeat step 24 for a total of 3 times to collect Amount600 µL   from the interlayer.

Dilute iodixanol with Amount600 µL   of Resuspension Buffer + Amount6 µL   of 7-AAD ready-made solution (Amount1 µL   stock concentration, Amount5 µL   final concentration). Pipet mix 10 times.

Centrifuge the tubes at Centrifigation500 x g, 4°C, 00:10:00 .

Note
If you start with a small amount of tissues, the pellet may not be visible after centrifugation. Therefore, it’s important to note the position the tubes in the centrifuge so that you know where the pellet is supposed to be after centrifugation. 

Note
Using a centrifuge with swinging bucket rotor will improve efficiency.

10m

Gently remove one tube at a time from the centrifuge. Remove the supernatant by inverting the tube. Leave behind Amount50 µL   of supernatant as not to disturb nuclei pellet.

Resuspend the pellets in each tube in Amount150 µL   of resuspension buffer. Pipet mix 10 times.

Nuclei Permeabilization

15m

Add Amount20 µL   of Nuclei Permeabilization Buffer to a final concentration of 0.1 X. Mix by inverting tube 5 times.

Incubate tubes TemperatureOn ice   for Duration00:03:00  .

Add Amount400 µL   Diluted Nuclei Buffer to dilute Nuclei Permeabilization Buffer and facilitate buffer exchange. Mix by inverting tube 5 times.

Centrifuge tubes at Centrifigation500 x g, 4°C, 00:10:00 . 

10m

Remove most of the supernatant without disrupting the nuclei pellet.

Centrifuge at  Centrifigation500 x g, 4°C, 00:02:00   to spin down excessive supernatant.

Carefully remove the supernatant without disturbing the pellet.

Nuclei count and dilution

Resuspend pellet in Amount10 µL   of chilled Diluted Nuclei Buffer. Pipet mix 10 time and pulse vortex for one second to resuspend the nuclei pellet.

For each sample, Dilute Amount1 µL   of nuclei in Amount11 µL   of Diluted Nuclei Buffer. Load Amount10 µL   on a hemocytometer.

Inspect and count on an epifluorescence microscope with a 7-AAD filter. 

Note
7-AAD has its peak excitation at ~550 nm and peak emission at ~650 nm.

A	B	C	D
NIM (Nuclear isolation media)	Stock	Final	100 mL
Nuclease-free Water			47.25 mL
Sucrose	0.5 M	0.25 M	50 mL
KCl	2 M	25 mM	1250 µL
MgCl2	1 M	5 mM	500 µL
Tris-HCl, pH 8.0	1 M	10 mM	1000 µL

A	B	C	D
Diluent	Stock	Final	50 mL
Nuclease-free Water			41.75 mL
KCl	2 M	150 mM	3.75 mL
MgCl2	1 M	30 mM	1.5 mL
Tris-HCl, pH 8.0	1 M	60 mM	3 mL

A	B	C	D
Homogenization Buffer	Stock	Final	1100µL per sample
NIM			1060 µL
BSA	30%	1%	37 µL
RNase inhibitor	40 U/ µL	0.08 U/ µL	2.2 µL

A	B	C	D
Resuspension Buffer	Stock	Final	1100µL per sample
PBS	1X	1X	1060 µL
BSA	30%	1%	37 µL
RNase inhibitor	40 U/ µL	0.08 U/ µL	2.2 µL

A	B	C	D
Nuclei Permeabilization Buffer	Stock	Final	1 ml per 8 samples
Nuclease-free Water			963 µL
Tris-HCl (pH 7.4)	1M	10 mM	10 µL
NaCl	5M	10 mM	2 µL
MgCl2	1M	3 mM	3 µL
Tween-20	10%	0.1%	10 µL
NP40	10%	0.1%	10 µL
Digitonin	5%	0.01%	2 µL

A	B	C	D
Diluted Nuclei Buffer	Stock	Final	55µL per sample
Nuclease-free Water	-	-	520 µL
Nuclei Buffer (20X)	20X	1X	27.5 µL
DTT	1M	1mM	0.55 µL
RNase inhibitor	40U/ µL	0.08 U/ µL	1.1 µL

	A	B
	50% iodixanol	3 mL per sample
	Opti-prep	2500 µL
	Diluent	500 µL

	A	B
	40% iodixanol	1100 µL per sample
	50% iodixanol	880 µL
	NIM	220 µL
	30% BSA	37 µL
	RNase inhibitor	2.2 µL

	A	B
	30% iodixanol	1100 µL per sample
	50% iodixanol	660 µL
	NIM	440 µL
	30% BSA	37 µL
	RNase inhibitor	2.2 µL

Public workspaceNuclear extraction using opti-prep gradient centrifugation for 10X Genomics Multiome Assay

Nuclear extraction using opti-prep gradient centrifugation for 10X Genomics Multiome Assay