Nuclei Preparation from Frozen Tissue for 10X Multiome using Dounce Homogenization, Iodixanol Gradient Centrifugation, and FANS (v1.2, Jan 2024)

Prepare stock Diluent Buffer (1 mL) and 50% iodixanol (6 mL) at room temperature, if needed.

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Diluent Buffer
Reagent	Stock Concentration	Final Concentration	1 mL
Tris-HCl, pH 8	1 M	120 mM	120 µL
KCl	2 M	150 mM	75 µL
MgCl2	1 M	30 mM	30 µL
Molecular biology water	-	-	775 µL

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50% Iodixanol
Reagent	Stock Concentration	Final Concentration	6 mL
OptiPrep Density Gradient Medium	60%	50%	5 mL
Diluent Buffer			1 mL

On ice Prepare all other buffers fresh on ice.

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NIM
Reagent	Stock Concentration	Final Concentration	Volume per Sample
Sucrose in water	1M	0.25M	1 mL
KCl	2M	25 mM	50 μl
MgCl2	1M	5 mM	20 μl
Tris-HCl, pH 7.5	1M	10 mM	40 μl
Molecular biology water	-	-	4 mL
TOTAL	-	-	5.114 mL

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NIM-DP
Reagent	Stock Concentration	Final Concentration	Volume per Sample
NIM buffer	1X	1X	4 mL
DTT in water	200 mM	1 mM	20 μl
Roche cOmplete, EDTA-free Protease Inhibitor Cocktail	25X	1X	160 μl
Recombinant RNasin	40 U/µL	1 U/ μl	100 μl
TOTAL	-	-	4.28 mL

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NIM-DP-L
Reagent	Stock Concentration	Final Concentration	Volume per Sample
NIM-DP	-	-	1.1 mL
IGEPAL CA-630	10%	0.1%	11 μl

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20% Iodixanol
Reagent	Stock Concentration	Final Concentration	2 mL
OptiPrep Density Gradient Medium	50%	20%	800 µL
NIM	-	-	1.2 mL
Roche cOmplete, EDTA-free Protease Inhibitor Cocktail	25X	1X	80 µL
DTT in water	200 mM	1 mM	10 µL
Recombinant RNasin	40 U/µL	1 U/µL	50 µL

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25% Iodixanol
Reagent	Stock Concentration	Final Concentration	1 mL
OptiPrep Density Gradient Medium	50%	25%	500 µL
NIM	-	-	500 µL
Roche cOmplete, EDTA-free Protease Inhibitor Cocktail	25X	1X	40 µL
DTT in water	200 mM	1 mM	5 µL
Recombinant RNasin	40 U/µL	1 U/µL	25 µL

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Sort Buffer (SB)
Reagent	Stock Concentration	Final Concentration	For 4 samples
Fatty acid-free BSA in PBS	10%	1%	200 µL
Roche cOmplete, EDTA-free Protease Inhibitor Cocktail	25X	1X	80 µL
7-AAD (in DMSO)	1 mM	2 µM	4 µL
Recombinant RNasin	40 U/µL	1 U/µL	50 µL
PBS	-	-	1666 µL
TOTAL	-	-	2000 µL

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Collection Buffer (CB)
Reagent	Stock Concentration	Final Concentration	For 4 samples
Fatty acid-free BSA in PBS	10%	5%	200 µL
Recombinant RNasin	40 U/µL	5 U/µL	50 µL
PBS	-	-	150 µL
TOTAL	-	-	400 µL

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Nuclear Permeabilization Buffer (NPB)
Reagent	Stock Concentration	Final Concentration	For 4 samples
Fatty acid-free BSA in PBS	-	5%	50 mg
IGEPAL-CA630	10%	0.2%	2 µL
DTT	200 mM	1 mM	5 µL
Roche cOmplete, EDTA-free Protease Inhibitor Cocktail	25X	1X	40 µL
Recombinant RNasin	40 U/µL	1 U/µL	25 µL
PBS			928 µL

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Wash Buffer (WB)
Reagent	Stock Concentration	Final Concentration	Volume per Sample
Fatty acid-free BSA in PBS	10%	1%	200 µL
Roche cOmplete, EDTA-free Protease Inhibitor Cocktail	25X	1X	80 µL
Tris-HCl, pH 7.5	1M	10 mM	20 µL
DTT	200 mM	1 mM	10 µL
MgCl2	1M	3 mM	6 µL
NaCl	5M	10 mM	4 µL
Tween-20	10%	0.01%	2 µL
Recombinant RNasin	40 U/µL	1 U/µL	50 µL
Molecular biology water	-	-	1628 µL

Pre-chill a large, swing-bucket tabletop centrifuge to 4°C.

Retrieve a 1 mL dounce homogenizer with 2 pestles (“Loose” and “Tight”) for each sample. Place on ice and allow to chill.

Add 1 mL of NIM-DP-L buffer to each dounce homogenizer.

• *If tissue mass is very small (< 50 mg), instead add 600 µL of NIM-DP-L.

Transfer each sample to a dounce homogenizer.

Using the loose pestle, gently homogenize the sample until most of the tissue has broken into small pieces (usually 5-10 strokes).

Switch to the tight pestle and homogenize until the solution is uniform with no obvious particles (usually 15-25 strokes). Be gentle and avoid introducing bubbles.

Transfer the full volume of homogenized sample to a 30 μm filter in a 1.5 mL Eppendorf Lobind tube.

Rinse each dounce with 1 mL of NIM-DP buffer and transfer the rinse to the filter.

Centrifuge for 10 mins at 1000 rcf, 4°C, and 3/3 acceleration/deceleration.

If tissue mass is very small (< 50 mg), skip steps 10-11

Discard the supernatant and resuspend the pellet in 1 mL of NIM-DP.

Centrifuge for 10 mins at 1000 rcf, 4°C, and 3/3 acceleration/deceleration.

Discard the supernatant and resuspend the pelleted nuclei in 2 mL of 20% iodixanol.

**If tissue mass is very small (< 50 mg), instead resuspend in 400 uL of 50% iodixanol (for a final iodixanol concentration of 20%).

Slowly pipette the suspension dropwise onto a 500 µL cushion of 25% iodixanol in a 5 mL Eppendorf Lobind tube. Do not mix this solution once transferred.

Centrifuge for 30 mins at 4000 rcf, 4C, and 3/1 acceleration/deceleration .

Discard the supernatant, leaving a small volume (< 20 µL) to avoid disturbing the pellet.

Resuspend the pellet in 500 µL of sort buffer and incubate on ice for 10 mins, protected from light.

Sort 120,000-130,000 nuclei into a 1.5 mL Eppendorf Lobind tube containing 90 μL of collection buffer using a Sony SH800 Cell Sorter.

Centrifuge the sorted nuclei for 5 mins at 500 rcf, 4°C, and 3/3 acceleration/deceleration.

Discard the supernatant.

Resuspend the pellet in 100 μL of NPB. Incubate on ice for 1 minute.

Add 900 μL of wash buffer.

Centrifuge for 5 mins at 500 rcf, 4°C, and 3/3 acceleration/deceleration.

Carefully remove the supernatant, leaving 10-15 μL to avoid disturbing the pellet.

Gently resuspend in 12 μL of 1X Nuclei Buffer (prepared from 10X Genomics Multiome protocol).

Stain an aliquot of nuclei with 0.4% Trypan Blue. Load 10 μL into one chamber of a hemocytometer.

Count nuclei in four quadrants. Average the count and determine the nuclei concentration (nuclei/μL).

Capture images from the microscope field at 10X and 20X magnification. Assess nuclei quality.

Follow the 10X Genomics protocol “Chromium Next GEM Single Cell Multiome ATAC + Gene Expression” (CG000338, Rev F) for the remainder of the experiment. Input 18,000 nuclei for each tagmentation reaction for a targeted recovery of ~10,000 nuclei.