Aug 02, 2024
Nuclei Isolation from Human Synovium and Infrapatellar Fat Pad for 10x Multiome
- Irene Lorenzo Gomez1,
- Erpei Wang1,
- Merissa Olmer1,
- Martin Lotz1
- 1Scripps Research
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Document Citation: Irene Lorenzo Gomez, Erpei Wang, Merissa Olmer, Martin Lotz 2024. Nuclei Isolation from Human Synovium and Infrapatellar Fat Pad for 10x Multiome. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov193molr2/v1
License: This is an open access document 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
Created: July 16, 2024
Last Modified: August 02, 2024
Document Integer ID: 103469
Keywords: Knee, Synovium, Fat Pad, IFP, IPFP, 10x Multiome, Single Nuclei, fresh frozen, RNA-seq, ATAC-seq
Funders Acknowledgements:
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Grant ID: AR082186 02
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Abstract
This protocol describes isolation of nuclei from fresh-frozen human knee synovium and fat pad for use in Omics analyses, including RNA-sequencing of ATAC-sequencing. Tissue dissociation was the most critical step in optimization which was monitored by nuclei yield, integrity and purity. Methods for quality control are also described.
Nuclei Isolation from Human Synovium and Infrapatellar Fat Pad for 10x Multiome
Materials
5-ml centrifuge tubes (Eppendorf, catalog number: 0030108310)
15-ml conical centrifuge tubes (BioPioneer, catalog number: CNT-15)
50-ml conical centrifuge tubes (Falcon, catalog number: 352098)
Cell strainers, 40-μm (Fisher, catalog number: 22363547)
Cell strainers, 20-μm (pluriSelect, catalog number: 43-50020-03)
Cell strainers, 10-μm (pluriSelect, catalog number: 43-50010-03)
Connector Ring (pluriSelect, catalog number: 41-50000-03)
Forceps
Scalpel (Fisher, catalog number: 72042-21
Petri dish (Fisher, catalog number: FB0875713)
Dry ice
Ultracentrifuge tubes (Beckman Coulter, catalog number: 344059)
RNase inhibitor (Sigma Aldrich, catalog number: 3335402001)
Recombinant Albumin solution (New England Biolabs, catalog number: B9200S)
PBS (Thermo Fisher Scientific, catalog number: 10010023)
DTT, Sigma-Aldrich, Catalog No. 646563
Tween 20 (Fisher, catalog number: BP337500)
Nonidet P40 Substitute (Sigma Aldrich, catalog number: 74385)
Acridine Orange/Propidium Iodide Stain (Logos Biosystems, catalog number: F23001)
Nuclei buffer (10x genomics)
Nuclei Isolation Cartridge, S2 Genomics, Part No. 100-063-732
Lysis Buffer (see Recipes)
Wash Buffer (see Recipes)
Diluent Buffer (see Recipes)
Singulator instrument and reagents:
Singulator S200 (S2 Genomics)
*We use a custom Low Volume Nuclei Isolation Protocol on Singulator
*Diluent buffer in Distilled Nuclease free water and sterile filtered before use (see Recipes)
Nuclei Isolation Protocol Using the Singulator S200
The Singulator S200 Automated Tissue Dissociation System (from S2 Genomics) is programmable and thus allows reproducible, operator-independent nuclei preparation. We use fresh snapfrozen tissues stored in Liquid Nitrogen.
1.0. The buffer recipes are prepared fresh for each nuclei experiment. These custom reagents are pre-chilled and loaded into the Singluator Single Shot holder. The Single Shot holder has two 15 ml conical tube slots for each of the S200 bays of the Singulator. One slot is labeled "Buffer" where we load the Washing Buffer, the other slot says "Enzyme" where we load the Lysis Buffer. Each 15 ml conical tube contains 3 ml of the buffers in their respective tubes.
Single Shot holder
1.1. Prior to loading 100-150mg of tissue into to the Singulator cartridge, it needs to be finely shaved and minced into small pieces using a scalpel. The tissue is minced on a petri dish kept on dry ice.
* Some tissues may become sticky when the temperature rises, so the petri dish to be used should also be cooled. Use LN if needed. Scalpel and forceps should be pre-cooled on dry ice to avoid thawing of the tissue fragments.
1.2. Transfer tissue fragments to a pre-cooled Singulator Nuclei Isolation Cartridge (kept at -20ºC), add 75 ul of RNase inhibitor (40 U/ul) directly into the disruption chamber of the Nuclei Isolation Cartridge. Using the custom Low Volume nuclei isolation program, the final volume in the cartridge is 3 ml, containing 3,000 units of RNase inhibitor total.
1.3. Run the Low Volume custom program on the Singulator:
1.4. Remove cartridge from Singulator, puncture the aluminum seal to collect the nuclei suspension. Pass this suspension through 20 μm and 10 μm strainers into a 50-ml LoBind conical tube on ice.
If the pestle is difficult to remove, due to negative pressure, let the pressure resolve and then remove the pestle. If some tissue homogenate is left in the tissue loading compartment, you can aspirate with a pipette and pass through a 40 μm strainer. Then combine this with the nuclei fraction that had passed through the 40 μm filter in the Singulator and pass through 20 μm and 10 μm strainers to remove tissue and cellular debris.
1.5. Transfer the flow-through to a 5 ml LoBind tube.
1.6. Centrifuge at 150 rcf for 10 min at 4°C.
1.7. Remove supernatant carefully and resuspend in 600μL PBS + Recombinant Albumin 0.02 mg/ml + 1U/μl RNase Inhibitor.
1.8. To remove cellular debris, centrifuge at 4℃, 150 rfc for 10 min to pellet the nuclei and retain the small debris in the supernatant.
1.9. Remove supernatant and resuspend nuclei pellet in 100 uL Diluted Nuclei Buffer and mix 18 uL of nuclei suspension with 2 uL of Acridine Orange.
1.10. Count nuclei using a Countess II FL Automated Cell Counter (AOPI staining).
Example of AOPI counts: Nuclei stained in RFP (orange), live cells and debris stain in GFP (green).
1.11. Nuclei should be visualized under a microscope at 40x or 60x magnification (AOPI staining) to assess nuclear membrane integrity, according to the nuclear integrity quality check guidance by 10x (https://10xgenomics.com).
A: High-quality nuclei have well-resolved edges. Optimal quality for single-cell gene expression libraries.
B: Mostly intact nuclei with minor evidence of blebbing. Quality single-cell gene expression libraries can still be produced.
C: Nuclei with strong evidence of blebbing. Proceed at your own risk.
D: Nuclei are no longer intact. Do not proceed!
1.12. Centrifuge at 150 rcf for 10 min at 4°C and remove the supernatant without disrupting the nuclei pellet.
1.13. Based on the nuclei concentration estimated at step 1.10, resuspend nuclei pellet in chilled Diluted Nuclei Buffer to a target concentration of 4,000 – 5,000 nuclei/ul, for a target nuclei recovery of 20,000 nuclei.
* Put only 5 μL into the PCR tube
1.14. Proceed immediately to Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide (CG000338) and minimize the time between nuclei preparation and chip loading.