TITLE: Chromium Nuclei Isolation for Single Cell Multiome ATAC + Gene Expression (CG000505)
AUTHORS: 10x Genomics

[DESCRIPTION]
The Chromium Nuclei Isolation Kit is an all-in-one solution for the
standardized isolation of nuclei from frozen tissue for use in
10x Genomics Single Cell assays. Frozen tissue samples are
homogenized with a pestle in Lysis Buffer and passed through a
column. Next, debris is removed via centrifugation in Debris Removal
Buffer. The isolated nuclei are then washed and resuspended and
loaded directly into compatible 10x Genomics Single Cell assays.

The Chromium Nuclei Isolation Kit streamlines the nuclei isolation
process into a single workflow, allowing for increased efficiency,
scalability through sample batching, and reduced experimental
variability using 10x Genomics pre-formulated reagents. The protocol is
designed to be compatible with a wide variety of tissue types and sizes.

This User Guide outlines the process for isolating Nuclei from frozen
tissues for use in compatible 10x Genomics Single Cell assays. Refer to
the Product Compatibility and Protocol Selector pages for additional
information on choosing the appropriate nuclei isolation kit and
protocol based on the intended downstream Single Cell assay.

[GUIDELINES]
Perform all steps at 4ºC

[STEPS]
SECTION: Nuclei Isolation: Tissue Dissociation
5. 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.
SECTION: Nuclei Isolation: Tissue Dissociation
6. Prepare Single Cell Multiome ATAC + Gene Expression buffers according to Buffer Preparation section and place on ice.
SECTION: Nuclei Isolation: Tissue Dissociation
7. Place Sample Dissociation Tube(s) on dry ice.
SECTION: Nuclei Isolation: Tissue Dissociation
8. Obtain frozen tissue sample(s) and place immediately on dry ice.
SECTION: Nuclei Isolation: Tissue Dissociation
9. Transfer frozen tissue (3–50 mg) to pre-chilled Sample Dissociation Tube.
SECTION: Nuclei Isolation: Tissue Dissociation
10. 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.
SECTION: Nuclei Isolation: Tissue Dissociation
11. 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.
SECTION: Nuclei Isolation: Tissue Dissociation
12. Incubate on ice for 10 min.
SECTION: Nuclei Isolation: Tissue Dissociation
13. 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.
SECTION: Nuclei Isolation: Tissue Dissociation
14. Centrifuge at 16,000 rcf for 20 sec at 4°C. See Tips & Best Practices on page 14 for centrifuge loading guidance.
SECTION: Nuclei Isolation: Tissue Dissociation
15. 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.
SECTION: Nuclei Isolation: Tissue Dissociation
16. 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.
SECTION: Nuclei Isolation: Isolation and cleanup
17. Resuspend nuclei pellet in 500 µl Debris Removal Buffer. Gently pipette mix at least 15x, continuing until no pellet can be visualized.
SECTION: Nuclei Isolation: Isolation and cleanup
18. 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.
SECTION: Nuclei Isolation: Isolation and cleanup
19. Resuspend nuclei pellet in 1 ml of Wash Buffer.
SECTION: Nuclei Isolation: Isolation and cleanup
20. 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.
SECTION: Nuclei Isolation: Isolation and cleanup
21. Resuspend nuclei pellet in 1 ml of Wash Buffer.
SECTION: Nuclei Isolation: Isolation and cleanup
22. 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.
SECTION: Nuclei Isolation: Isolation and cleanup
23. 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.
SECTION: Nuclei Isolation: Isolation and cleanup
24. 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.
SECTION: Nuclei Isolation: Isolation and cleanup
25. 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.
SECTION: Nuclei Isolation: Isolation and cleanup
26. 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.
SECTION: Nuclei Isolation: Isolation and cleanup
27. Keep samples on ice and proceed immediately to relevant 10x Genomics User Guide.
SECTION: Buffer Preparation
1. Prepare Lysis Buffer (500 μL/rxn)


 

SECTION: Buffer Preparation
2. Prepare Debris Removal Buffer (500 μL/rxn)

 

SECTION: Buffer Preparation
3. Prepare Wash Buffer (2 mL/rxn)

 


SECTION: Buffer Preparation
4. Prepare Resuspension Buffer (1 mL/rxn)