Aug 12, 2022
Chromium Nuclei Isolation for Single Cell Multiome ATAC + Gene Expression (CG000505)
- 10x Genomics1
- 110x Genomics
Protocol Citation: 10x Genomics 2022. Chromium Nuclei Isolation for Single Cell Multiome ATAC + Gene Expression (CG000505). protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj65rwlk5/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: August 12, 2022
Last Modified: August 12, 2022
Protocol Integer ID: 68587
Keywords: scRNA-seq, scATAC-seq, 10x Genomics, nuclei, nuclei isolation
Abstract
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
Materials
1X Phospate-Buffered Saline without Ca2 and Mg2 CorningCatalog #21-040-CV
10% Bovine Serium AlbuminMerck Millipore SigmaCatalog #A1595
RNase Inhibitor10x GenomicsCatalog #2000565
20X Nuclei buffer10x GenomicsCatalog #2000207
Reducing Agent B10x GenomicsCatalog #2000087
Molecular Grade Nuclease-free WaterThermo Fisher ScientificCatalog #AM9937
Lysis Reagent10x GenomicsCatalog #2000558 Surfactant A10x GenomicsCatalog #2000559 Pestle10x GenomicsCatalog #2000561 Nuclei Isolation column10x GenomicsCatalog #2000562 Collection Tube10x GenomicsCatalog #2000563 Sample Dissociation Tube10x GenomicsCatalog #2000564
DNA LoBind Tube 2.0 mLEppendorfCatalog #022431048 15 mL centrifuge tubeCorningCatalog #CLS430791 50 mL centrifuge tubeCorningCatalog #CLS430829 VitaStain AOPI Staining SolutionNexcelomCatalog #CS2-0106-5ml
Equipment
Cellaca MX High-throughput Automated Cell Counter
NAME
cell counter
TYPE
Nexcelom
BRAND
MX0112-0127
SKU
Equipment
VWR ANALOG VORTEX MIXER
NAME
VWR
BRAND
10153-838
SKU
With tube insert
SPECIFICATIONS
Equipment
Centrifuge
NAME
Bench centrifuge
TYPE
Eppendorf
BRAND
5424
SKU
Safety warnings
None
Buffer Preparation
Buffer Preparation
10m
10m
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 |
10m
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 |
Nuclei Isolation: Tissue Dissociation
Nuclei Isolation: Tissue Dissociation
10m
10m
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.
Nuclei Isolation: Isolation and cleanup
Nuclei Isolation: Isolation and cleanup
45m
45m
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.