Prepare buffers fresh and leave on ice.

The volume of MACS Buffer prepared depends on the input mass of each sample. An additional 1 mL should be prepared for rinsing in addition to the homogenization volume. Refer to the chart below:

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A	B	C	D
MACS Buffer
Reagent	Stock Concentration	Final Concentration	for 1 mL
Roche cOmplete, EDTA-free Protease Inhibitor Cocktail	25X	1X	40 μl
DTT	200 mM	0.6 mM	3 μl
CaCl2	250 mM	5 mM	20 μl
EDTA	500 mM	5 mM	10 μl
Tris-HCl, pH 8.0	1M	10 mM	10 μl
MgAc	300 mM	3 mM	10 μl
Recombinant RNasin	40 U/µl	1 U/µl	25 μl
Molecular biology water	-	-	1.1 mL

A	B	C	D
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 (10% in DMSO)	1 mM	2 µM	4 µL
Recombinant RNasin	40 U/µl	1 U/µl	50 µL
PBS			1666 µL

A	B	C	D
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

A	B	C	D
Nuclear Permeabilization Buffer (NPB)
Reagent	Stock Concentration	Final Concentration	1 mL
Fatty acid-free BSA in PBS	-	5%	50 mg
IGEPAL-CA630	10%	0.20%	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

A	B	C	D
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.

Add the pre-determined homogenization volume of MACS buffer to each MACS tube on ice.

Immediately transfer samples to MACS tubes. If needed, resuspend tissue first with MACS buffer from the MACS tube and then transfer the full volume to the tube.

Tighten the cap of each MACS tube and flip the tube upside down. Ensure that all tissue is submerged in the buffer.

Thaw on ice for 1 min.

Homogenize samples using the gentleMACS Octo Dissociator in the 4 °C cold room.

Run the protocol “protein_01_01” for gentleMACS M tubes (~1 min).

Quick spin M tubes to bring all liquid to the bottom of the tubes.

Filter each sample into a 15 mL tube using a 30 um (green) Celltrics filter.

Rinse the sides of each MACS tube with 1 mL MACS buffer.

Quick spin M tubes to bring all liquid to the bottom of the tubes.

Transfer the rinse to each Celltrics filter.

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

Discard supernatants. A small volume of supernatant can be left in each tube to avoid disturbing the pellet.

Gently resuspend each pellet in 500 µL sort buffer.

Transfer the full sample volume to a pre-chilled 1.5 mL LoBind tube.

Incubate on ice, protected from light, for 10 min.

Aliquot 90 uL of 5X collection buffer into a 1.5 mL LoBind tube for each sample.

Sort 120,000 nuclei into the 90 µL of collection buffer for each sample.

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

Discard supernatant.

Gently resuspend pellet in 100 µL of NPB.

Incubate on ice for 1 min.

Add 900 µL of Wash buffer to each sample

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

Carefully discard supernatants. Switch to a P20 pipette once the volume reaches ~40 µL. Do NOT disturb the pellet.

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

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.