Chromium Nuclei Isolation for Single Cell Multiome ATAC + Gene Expression (CG000505)

Prepare Lysis Buffer (500 μL/rxn)

A	B	C	D	E
	PN	1X + 10% (μL)	4X + 10% (μL)	8X + 10% (μL)
Lysis Reagent	2000558	550	2200	4400
Reducing Agent B	2000087	0.55	2.2	4.4
Surfactant A	2000559	5.5	22	44
Total		556.05	2224.2	4448.4

Prepare Debris Removal Buffer (500 μL/rxn)

A	B	C	D	E
	PN	1X + 10% (μL)	4X + 10% (μL)	8X + 10% (μL)
Debris Removal Reagent	2000560	550	2200	4400
Reducing Agent B	2000087	0.55	2.2	4.4
Total		550.55	2202.2	4404.4

Prepare Wash Buffer (2 mL/rxn)

A	B	C	D	E
	PN	1X + 10% (μL)	4X + 10% (μL)	8X + 10% (μL)
1X PBS	-	1925	7700	15400
10% BSA	-	220	880	1760
RNase Inhibitor	2000565	55	220	440
Total		2200	8800	1760

Prepare Resuspension Buffer (1 mL/rxn)

A	B	C	D	E
	PN	1X + 10% (μL)	4X + 10% (μL)	8X + 10% (μL)
20X Nuclei Buffer	2000207	55	220	440
Reducing Agent B	2000087	1.1	4.4	8.8
Nuclease-free Water	-	1016	4066	8131
RNase Inhibitor	2000565	27.5	110	220
Total		1099.6	4400.4	8799.8

Pre-chill centrifuge to 4°C and place reagents and tubes on ice as indicated in the Get Started guide. Label tops and sides of tubes, as well as tops of spin columns, before starting protocol. Perform all protocol steps on ice and centrifugation steps at 4°C.

Prepare Single Cell Multiome ATAC + Gene Expression buffers according to Buffer Preparation section and place on ice.

Place Sample Dissociation Tube(s) on dry ice.

Obtain frozen tissue sample(s) and place immediately on dry ice.

Transfer frozen tissue (3–50 mg) to pre-chilled Sample Dissociation Tube.

Transfer Sample Dissociation Tubes(s) to wet ice. Add 200 µl Lysis Buffer to Sample Dissociation Tube. Dissociate tissue with plastic pestle until homogeneous. For multiple samples, add Lysis Buffer to each tissue and then proceed to dissociate one at a time. Perform tissue dissociation on ice. Use one pestle per sample. DO NOT discard pestles until nuclei isolation process is complete.

Add 300 µl Lysis Buffer. Pipette mix 10x. If pipette tip clogs with unhomogenized tissue, continue to dissociate tissue with the pestle until able to pipette mix.

Incubate on ice for 10 min.

Pipette dissociated tissue into pre-chilled Nuclei Isolation Column assembled with Collection Tube using pipette set to 500 μl. Transfer all liquid from Dissociation Tube to Nuclei Isolation Column to avoid nuclei loss.

Centrifuge at 16,000 rcf for 20 sec at 4°C. See Tips & Best Practices on page 14 for centrifuge loading guidance.

Discard column. Flowthrough in the Collection Tube will contain nuclei. Vortex 10 sec at 3,200 rpm or max speed to resuspend nuclei. Flowthrough may appear opaque or cloudy. This is normal and it is safe to proceed.

Centrifuge Collection Tube for 3 min at 500 rcf at 4°C. Carefully discard supernatant using a pipette without disturbing nuclei pellet. Leave behind a small fraction (~200 µl) of supernatant if nuclei pellet is not apparent. Position tubes with hinges facing in same direction within the centrifuge, which ensures that the pellet is consistently in the same place (opposite the hinge) following centrifugation.

Resuspend nuclei pellet in 500 µl Debris Removal Buffer. Gently pipette mix at least 15x, continuing until no pellet can be visualized.

Centrifuge at 700 rcf for 10 min at 4°C. Carefully discard supernatant using a pipette without disturbing nuclei pellet. Leave behind a small fraction (~200 µl) of supernatant if nuclei pellet is not apparent.

Resuspend nuclei pellet in 1 ml of Wash Buffer.

Centrifuge at 500 rcf for 5 min at 4°C. Carefully discard supernatant using a pipette without disturbing nuclei pellet. Leave behind a small fraction (~200 μl) of supernatant if nuclei pellet is not apparent.

Resuspend nuclei pellet in 1 ml of Wash Buffer.

Centrifuge at 500 rcf for 5 min at 4°C. Carefully discard as much supernatant as possible using a pipette without disturbing nuclei pellet. Leave behind a small remaining volume if the pellet is not visible.

Resuspend nuclei pellet in 50–500 µl Resuspension Buffer, depending on expected recovery for input tissue type and mass. Refer to Nuclei Recovery section of Tips & Best Practices for information on typical nuclei recovery. Gently pipette mix 15x using an appropriate pipette for resuspension volume.

Vortex nuclei for 3 sec at 3,200 rpm or max speed immediately prior to counting to ensure accurate nuclei count. Pulse spin the tube after vortexing to collect liquid at bottom of tube. DO NOT pulse spin the tube for more than 1 second to ensure that nuclei do not pellet at the bottom of the tube.

Determine nuclei concentration using AOPI or Ethidium Homodimer-1 fluorescent staining dyes and dilute if necessary for target nuclei load. Follow recommendations for nuclei counting as outlined in the Tips & Best Practices on page 19 of this document. Adjust nuclei concentration as necessary for intended downstream assay.

Vortex nuclei for 3 sec at 3,200 rpm or max speed. Pulse spin the tube after vortexing to collect liquid at bottom of tube. DO NOT pulse spin the tube for more than 1 second to ensure that nuclei do not pellet at the bottom of the tube.

Keep samples on ice and proceed immediately to relevant 10x Genomics User Guide.