Feb 24, 2024
Nuclei Preparation from Frozen Tissue for 10X Multiome using Dounce Homogenization, Iodixanol Gradient Centrifugation, and FANS (v1.2, Jan 2024)
- 1Center for Epigenomics
Protocol Citation: Emily Eisner 2024. Nuclei Preparation from Frozen Tissue for 10X Multiome using Dounce Homogenization, Iodixanol Gradient Centrifugation, and FANS (v1.2, Jan 2024). protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzx435gx1/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: February 24, 2024
Last Modified: February 24, 2024
Protocol Integer ID: 95698
Keywords: Multiome, Dounce homogenization, Gradient Centrifugation, FANS, Nuclei Isolation
Abstract
This protocol describes isolation of nuclei from frozen tissue using dounce homogenization, iodixanol gradient centrifugation, and FANS. Nuclei are permeabilized, washed, and counted; single-nucleus suspensions of sufficient concentration and nuclei quality may then be processed using the Chromium Next GEM Single Cell Multiome ATAC + Gene Expression (CG000338, Rev F) protocol from 10X Genomics.
Attachments
Materials
Reagents List:
| A | B | C | D | |
| Reagent | Concentration | Vendor | Catalog Number | |
| Sucrose | - | Sigma | S1888-500G | |
| KCl | 1M | Invitrogen | AM9640G | |
| MgCl2 | 1M | Invitrogen | AM9530G | |
| Tris-HCl, pH 7.5 | 1M | Invitrogen | 15567-027 | |
| Tris-HCl, pH 8.0 | 1M | Invitrogen | 15568-025 | |
| DTT (DL-Dithiothreitol) | - | Sigma | D9779-10G | |
| Roche cOmplete, EDTA-free Protease Inhibitor Cocktail Tablets | - | Sigma | 5056489001 | |
| Recombinant RNasin (Ribonuclease Inhibitor), 10000 U | - | Promega | N2515 | |
| Molecular biology water | - | Corning | 46-000-CV | |
| IGEPAL CA-630 | - | Sigma | I8896-50ML | |
| Tween-20 | 10% | BioRad | 1662404 | |
| NaCl | 5M | Invitrogen | AM9760G | |
| OptiPrep Density Gradient Medium (Iodixanol) | - | Sigma | D1556-250ML | |
| Fatty acid-free BSA | - | Lampire Biological Laboratories | 7500804 | |
| 7-AAD | - | Invitrogen | A1310 | |
| PBS | - | Corning | 21-040-CV | |
| Trypan Blue | 0.4% | Invitrogen | T10282 |
Equipment:
Sony Cell Sorter (SH800)
Eppendorf tabletop swing-bucket centrifuge (Eppendorf, 5920R)
Consumables
Wheaton Dounce Tissue Grinder, 1 mL (DWK Life Sciences, 357538)
Sony Sorting Chip-100 µm for SH800 and MA900 (Sony, LEC3210)
Thermo Scientific‱ NERL‱ Diluent 2 Hematology Reagent for Flow Cytometry (Fisher Scientific, 23-029-361)
30 μm CellTrics (Fisher Scientific, NC9682496)
1.5 mL Lo Bind Centrifuge tubes (Eppendorf, 022431021)
5 mL Eppendorf DNA LoBind tubes (Eppendorf, 0030108310)
Thermo Scientific‱ SoftFit-L‱ Filtered Pipette Tips in Hinged Racks, 200 µL (Fisher Scientific, 21-402-561)
Thermo Scientific‱ SoftFit-L‱ Filtered Pipette Tips in Hinged Racks, 20 µL (Fisher Scientific, 21-402-550)
xTIP4‱ Racked Pipette Tips, Rainin‱ LTS‱ Pipette Compatible, Biotix, 1000 µL (Fisher Scientific, 76266-146)
Olympus Plastics 0.2 mL 8-Strip PCR Tubes, Flex Free Individual Attached Flat Caps (Genesee Scientific, 27-125U)
Serological Pipets, 10 mL, Sterile, Individually Wrapped (Genesee Scientific, 12-104)
Reagent Preparation
Reagent Preparation
Prepare stock Diluent Buffer (1 mL) and 50% iodixanol (6 mL) at room temperature, if needed.
| A | B | C | D | |
| A | B | C | D | |
| Diluent Buffer | ||||
| Reagent | Stock Concentration | Final Concentration | 1 mL | |
| Tris-HCl, pH 8 | 1 M | 120 mM | 120 µL | |
| KCl | 2 M | 150 mM | 75 µL | |
| MgCl2 | 1 M | 30 mM | 30 µL | |
| Molecular biology water | - | - | 775 µL | |
| A | B | C | D | |
| 50% Iodixanol | ||||
| Reagent | Stock Concentration | Final Concentration | 6 mL | |
| OptiPrep Density Gradient Medium | 60% | 50% | 5 mL | |
| Diluent Buffer | 1 mL | |||
On ice Prepare all other buffers fresh on ice.
| A | B | C | D | |
| NIM | ||||
| Reagent | Stock Concentration | Final Concentration | Volume per Sample | |
| Sucrose in water | 1M | 0.25M | 1 mL | |
| KCl | 2M | 25 mM | 50 μl | |
| MgCl2 | 1M | 5 mM | 20 μl | |
| Tris-HCl, pH 7.5 | 1M | 10 mM | 40 μl | |
| Molecular biology water | - | - | 4 mL | |
| TOTAL | - | - | 5.114 mL | |
| A | B | C | D | |
| NIM-DP | ||||
| Reagent | Stock Concentration | Final Concentration | Volume per Sample | |
| NIM buffer | 1X | 1X | 4 mL | |
| DTT in water | 200 mM | 1 mM | 20 μl | |
| Roche cOmplete, EDTA-free Protease Inhibitor Cocktail | 25X | 1X | 160 μl | |
| Recombinant RNasin | 40 U/µL | 1 U/ μl | 100 μl | |
| TOTAL | - | - | 4.28 mL | |
| A | B | C | D | |
| NIM-DP-L | ||||
| Reagent | Stock Concentration | Final Concentration | Volume per Sample | |
| NIM-DP | - | - | 1.1 mL | |
| IGEPAL CA-630 | 10% | 0.1% | 11 μl | |
| A | B | C | D | |
| 20% Iodixanol | ||||
| Reagent | Stock Concentration | Final Concentration | 2 mL | |
| OptiPrep Density Gradient Medium | 50% | 20% | 800 µL | |
| NIM | - | - | 1.2 mL | |
| Roche cOmplete, EDTA-free Protease Inhibitor Cocktail | 25X | 1X | 80 µL | |
| DTT in water | 200 mM | 1 mM | 10 µL | |
| Recombinant RNasin | 40 U/µL | 1 U/µL | 50 µL | |
| A | B | C | D | |
| 25% Iodixanol | ||||
| Reagent | Stock Concentration | Final Concentration | 1 mL | |
| OptiPrep Density Gradient Medium | 50% | 25% | 500 µL | |
| NIM | - | - | 500 µL | |
| Roche cOmplete, EDTA-free Protease Inhibitor Cocktail | 25X | 1X | 40 µL | |
| DTT in water | 200 mM | 1 mM | 5 µL | |
| Recombinant RNasin | 40 U/µL | 1 U/µL | 25 µL | |
| 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 (in DMSO) | 1 mM | 2 µM | 4 µL | |
| Recombinant RNasin | 40 U/µL | 1 U/µL | 50 µL | |
| PBS | - | - | 1666 µL | |
| TOTAL | - | - | 2000 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 | |
| TOTAL | - | - | 400 µL | |
| A | B | C | D | |
| Nuclear Permeabilization Buffer (NPB) | ||||
| Reagent | Stock Concentration | Final Concentration | For 4 samples | |
| Fatty acid-free BSA in PBS | - | 5% | 50 mg | |
| IGEPAL-CA630 | 10% | 0.2% | 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 | |
Nuclei Preparation
Nuclei Preparation
Pre-chill a large, swing-bucket tabletop centrifuge to 4°C.
Retrieve a 1 mL dounce homogenizer with 2 pestles (“Loose” and “Tight”) for each sample. Place on ice and allow to chill.
Add 1 mL of NIM-DP-L buffer to each dounce homogenizer.
**If tissue mass is very small (< 50 mg), instead add 600 µL of NIM-DP-L.
Transfer each sample to a dounce homogenizer.
Using the loose pestle, gently homogenize the sample until most of the tissue has broken into small pieces (usually 5-10 strokes).
Switch to the tight pestle and homogenize until the solution is uniform with no obvious particles (usually 15-25 strokes). Be gentle and avoid introducing bubbles.
Transfer the full volume of homogenized sample to a 30 μm filter in a 1.5 mL Eppendorf Lobind tube.
Rinse each dounce with 1 mL of NIM-DP buffer and transfer the rinse to the filter.
Centrifuge for 10 mins at 1000 rcf, 4°C, and 3/3 acceleration/deceleration.
**If tissue mass is very small (< 50 mg), skip steps 10-11**
Discard the supernatant and resuspend the pellet in 1 mL of NIM-DP.
Centrifuge for 10 mins at 1000 rcf, 4°C, and 3/3 acceleration/deceleration.
Discard the supernatant and resuspend the pelleted nuclei in 2 mL of 20% iodixanol.
**If tissue mass is very small (< 50 mg), instead resuspend in 400 uL of 50% iodixanol (for a final iodixanol concentration of 20%).
Slowly pipette the suspension dropwise onto a 500 µL cushion of 25% iodixanol in a 5 mL Eppendorf Lobind tube. **Do not mix this solution once transferred.**
Centrifuge for 30 mins at 4000 rcf, 4C, and 3/1 acceleration/deceleration.
Discard the supernatant, leaving a small volume (< 20 µL) to avoid disturbing the pellet.
Resuspend the pellet in 500 µL of sort buffer and incubate on ice for 10 mins, protected from light.
Sort 120,000-130,000 nuclei into a 1.5 mL Eppendorf Lobind tube containing 90 μL of collection buffer using a Sony SH800 Cell Sorter.
Centrifuge the sorted nuclei for 5 mins at 500 rcf, 4°C, and 3/3 acceleration/deceleration.
Discard the supernatant.
Resuspend the pellet in 100 μL of NPB. Incubate on ice for 1 minute.
Add 900 μL of wash buffer.
Centrifuge for 5 mins at 500 rcf, 4°C, and 3/3 acceleration/deceleration.
Carefully remove the supernatant, leaving 10-15 μL to avoid disturbing the pellet.
Gently resuspend in 12 μL of 1X Nuclei Buffer (prepared from 10X Genomics Multiome 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. Assess nuclei quality.
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.