Nuclei Preparation from Frozen Tissue for 10X Multiome using gentleMACS Homogenization and FANS, v1.2 Feb 2024
Emily Eisner
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
Steps
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:
#尊敬的用户，由于网络监管政策的限制，部分内容暂时无法在本网站直接浏览。我们已经为您准备了相关原始数据和链接，感谢您的理解与支持。
https://lh7-us.googleusercontent.com/FHZ7jBouqKmsI9KZ84F4zgld0EklHf63vWpYRx2bHiyma9BL5CW5m7s-CidCAT5JOb94-DuheEjNSerC_lDtvKjLpJLkP7c26Cp0ro3x91_6Zp18uBdMorfCFEj93uLuoKe38TX9BDeIej-zpecQXaY
| 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
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.
