Feb 24, 2024
Nuclei Preparation from Frozen Tissue for 10X Multiome using gentleMACS Homogenization and FANS, v1.2 Feb 2024
- 1Center for Epigenomics, University of California, San Diego
Protocol Citation: Emily Eisner 2024. Nuclei Preparation from Frozen Tissue for 10X Multiome using gentleMACS Homogenization and FANS, v1.2 Feb 2024. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn35zml5d/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: 95699
Keywords: Nuclei isolation, FANS, gentleMACS, Tissue homogenization, Multiome
Abstract
This protocol describes isolation of nuclei from frozen tissue using gentleMACS homogenization 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 | |
| CaCl2 | 1M | Thermo Scientific | J63122.AE | |
| EDTA, pH 8.0 | 0.5M | Invitrogen | AM9260G | |
| MgCl2 | 1M | Invitrogen | AM9530G | |
| Mg acetate (MgAc) | 1M | Thermo Scientific | J60041.AD | |
| 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 | |
| Fatty acid-free BSA | - | Lampire Biological Laboratories | 7500804 | |
| 7-AAD | - | Invitrogen | A1310 | |
| PBS | - | Corning | 21-040-CV | |
| Trypan Blue | 0.4% | Invitrogen | T10282 | |
| DMSO (Dimethyl Sulfoxide) | - | MP Biomedicals | ICN19481980 |
Equipment:
Sony Cell Sorter (SH800)
Eppendorf tabletop swing-bucket centrifuge (Eppendorf, 5920R)
gentleMACS Octo Dissociator with Heaters (Miltenyi Biotec)
Consumables:
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)
gentleMACS M tubes, sterile packed as 4x25 pieces (Miltenyi Biotec,130-096-335)
Standard Line Sterile Centrifuge Tubes with Flat Caps, Conical-Bottom, 15 mL (VWR, 10025-686)
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 buffers fresh and leave on ice.
The volume of MACS Buffer prepared depends on the input mass of each sample. An additional 1 mL should be prepared for rinsing in addition to the homogenization volume. Refer to the chart below:
| A | B | C | D | |
| MACS Buffer | ||||
| Reagent | Stock Concentration | Final Concentration | for 1 mL | |
| Roche cOmplete, EDTA-free Protease Inhibitor Cocktail | 25X | 1X | 40 μl | |
| DTT | 200 mM | 0.6 mM | 3 μl | |
| CaCl2 | 250 mM | 5 mM | 20 μl | |
| EDTA | 500 mM | 5 mM | 10 μl | |
| Tris-HCl, pH 8.0 | 1M | 10 mM | 10 μl | |
| MgAc | 300 mM | 3 mM | 10 μl | |
| Recombinant RNasin | 40 U/µl | 1 U/µl | 25 μl | |
| Molecular biology water | - | - | 1.1 mL | |
| 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 (10% in DMSO) | 1 mM | 2 µM | 4 µL | |
| Recombinant RNasin | 40 U/µl | 1 U/µl | 50 µL | |
| PBS | 1666 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 | |
| A | B | C | D | |
| Nuclear Permeabilization Buffer (NPB) | ||||
| Reagent | Stock Concentration | Final Concentration | 1 mL | |
| Fatty acid-free BSA in PBS | - | 5% | 50 mg | |
| IGEPAL-CA630 | 10% | 0.20% | 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.
Add the pre-determined homogenization volume of MACS buffer to each MACS tube on ice.
Immediately transfer samples to MACS tubes. If needed, resuspend tissue first with MACS buffer from the MACS tube and then transfer the full volume to the tube.
Tighten the cap of each MACS tube and flip the tube upside down. Ensure that all tissue is submerged in the buffer.
Thaw on ice for 1 min.
Homogenize samples using the gentleMACS Octo Dissociator in the 4 °C cold room.
Run the protocol “protein_01_01” for gentleMACS M tubes (~1 min).
Quick spin M tubes to bring all liquid to the bottom of the tubes.
Filter each sample into a 15 mL tube using a 30 um (green) Celltrics filter.
Rinse the sides of each MACS tube with 1 mL MACS buffer.
Quick spin M tubes to bring all liquid to the bottom of the tubes.
Transfer the rinse to each Celltrics filter.
Centrifuge homogenized samples for 5 min at 500 rcf, 4°C, and 3/3 acceleration/deceleration.
Discard supernatants. A small volume of supernatant can be left in each tube to avoid disturbing the pellet.
Gently resuspend each pellet in 500 µL sort buffer.
Transfer the full sample volume to a pre-chilled 1.5 mL LoBind tube.
Incubate on ice, protected from light, for 10 min.
Aliquot 90 uL of 5X collection buffer into a 1.5 mL LoBind tube for each sample.
Sort 120,000 nuclei into the 90 µL of collection buffer for each sample.
Centrifuge for 5 min at 500 rcf, 4°C, and 3/3 acceleration/deceleration.
Discard supernatant.
Gently resuspend pellet in 100 µL of NPB.
Incubate on ice for 1 min.
Add 900 µL of Wash buffer to each sample
Centrifuge for 5 min at 500 rcf, 4°C, and 3/3 acceleration/deceleration.
Carefully discard supernatants. Switch to a P20 pipette once the volume reaches ~40 µL. Do NOT disturb the pellet.
Gently resuspend in 12 μL of 1X Nuclei Buffer (prepared from 10X Genomics 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.
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.