Dec 17, 2021
Version 2
Isolation of nuclei from frozen human skeletal and cardiac muscle for single nucleus RNA and chromatin assays V.2
- James R Hocker1,
- Sebastian Preissl1,
- Dinh H Diep1
- 1University of California, San Diego
Protocol Citation: James R Hocker, Sebastian Preissl, Dinh H Diep 2021. Isolation of nuclei from frozen human skeletal and cardiac muscle for single nucleus RNA and chromatin assays . protocols.io https://dx.doi.org/10.17504/protocols.io.b2z4qf8wVersion created by Dinh H Diep
Manuscript citation:
Hocker, J. D., Poirion, O. B., Zhu, F., Buchanan, J., Zhang, K., Chiou, J., Wang, T.-M., Zhang, Q., Hou, X., Li, Y. E., Zhang, Y., Farah, E. N., Wang, A., McCulloch, A. D., Gaulton, K. J., Ren, B., Chi, N. C., & Preissl, S. (2021). Cardiac cell type–specific gene regulatory programs and disease risk association. Science Advances, 7(20), eabf1444. https://doi.org/10.1126/sciadv.abf1444
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: December 17, 2021
Last Modified: December 17, 2021
Protocol Integer ID: 56092
Abstract
This protocol was adapted for the isolation of single nuclei from frozen skeletal and cardiac muscle tissues for molecular characterization with the SNARE-seq2, sci-ATAC-seq, and snRNA-seq assays.
References:
(1) Preissl et al (2015). Circulation Research. Doi: 10.1161/CIRCRESAHA.115.306337
(2) Hocker et al (2021). Science Advances. Doi: 10.1126/sciadv.abf1444
Materials
Required consumables per sample:
- gentleMACS M Tube (Miltenyi Biotec, 130-096-335)
- CellTrics filter, 30 uM (CellTrics, 04-0042-2316)
- Eppendorf Tubes, 5 mL
Required instrument:
- gentleMACS Tissue Dissociator (Miltenyi Biotec, 130-095-937)
Buffers:
| A | B | C | D | E | F | |
| Reagent | Stock conc. | Final conc. | 1 mL | 5 mL | 10 mL | |
| PBS | 1X | 1X | 0.925 mL | 4.625 mL | 9.25 mL | |
| BSA (Sigma Aldrich) | n/a | 5% | 50 mg | 250 mg | 500 mg | |
| IGEPAL CA-630 / NP 40 | 10% | 0.20% | 20 uL | 100 uL | 200 uL | |
| DTT | 100 mM | 1 mM | 10 uL | 50 uL | 100 uL | |
| Protease Inhibitor | 25X | 1X | 40 uL | 200 uL | 400 uL | |
| Enzymatics RNase In | 40 U/uL | 0.2 U/uL | 5 uL | 25 uL | 50 uL |
Nuclear Permeabilization Buffer (NPB). Make fresh for each use.
| A | B | C | D | E | F | |
| Reagent | Stock conc. | Final conc. | 1 mL | 4 mL | 12 mL | |
| Nuclease free water | n/a | n/a | 0.927 mL | 3.708 mL | 11.124 mL | |
| CaCl2 | 1 M | 5 mM | 5 uL | 20 uL | 60 uL | |
| MgOAC | 1 M | 3 mM | 3 uL | 12 uL | 36 uL | |
| Tris-HCl, pH 8.0 | 1 M | 10 mM | 10 uL | 40 uL | 120 uL | |
| EDTA | 500 mM | 2 mM | 4 uL | 16 uL | 48 uL | |
| DTT | 100 mM | 0.6 mM | 6 uL | 24 uL | 72 uL | |
| Protease Inhibitor | 25X | 1X | 40 uL | 160 uL | 480 uL | |
| Enzymatics RNase In | 40 U/uL | 0.2 U/uL | 5 uL | 20 uL | 60 uL |
MACS Buffer. Make a stock without Protease In and add when using.
| A | B | C | D | E | F | |
| Reagent | Stock conc. | Final conc. | 1 mL | 5 mL | 10 mL | |
| PBS | 1X | 1X | 0.93 mL | 4.65 mL | 9.3 mL | |
| Superase In | 20 U/uL | 0.05 U/uL | 2.5 uL | 12.5 uL | 25 uL | |
| Enzymatics RNase In | 40 U/uL | 0.05 U/uL | 1.25 uL | 6.75 uL | 12.5 uL |
PBS + Rnase In. Make fresh for each use.
Section flash-frozen skeletal muscle into aliquots according to desired nuclei yield and store on dry ice or at -80 °C .
Note
Expect yield of 2000-4000 nuclei per mg of skeletal muscle tissue.
Prepare buffers and chill components:
| A | B | C | |
| Tissue mass | MACS buffer | NPB buffer | |
| 10-50mg | 1 mL | 2 mL | |
| 50-100mg | 2 mL | 4 mL | |
| >100mg | 3 mL | 1 mL per 25 mg |
Note: May require further optimization.
- Prepare buffer fresh on day of nuclei isolation
- Precool centrifuge to 4 °C
- Precool all buffers at 4 °C
- Place gentleMACS dissociator in cold room / chiller
- Chill gentleMACS M tubes on ice
- Chill 5.0 mL Eppendorf tubes on ice
Transfer sectioned frozen tissue to gentleMACS M tubes.
Add the recommended volume of MACS buffer to gentleMACS M tube.
Note
Allow tissue to thaw in MACS buffer for approximately 60 seconds on ice.
Homogenize with Miltenyi tissue dissociation protocol: "Protein_01_01" on the gentleMACS instrument in the chiller/cold room.
Briefly centrifuge the gentleMACS M tube to pull all the homogenate to the bottom.160 x g, 4°C, 00:00:15
15s
Filter homogenate through 30 uM CellTrics filter into 5 mL Eppendorf Tubes.
Wash gentleMACS M tube with another 1 mL of MACS buffer and filter the wash.
900 x g, 4°C, 00:10:00
Note
Use ramp rate: 3/9 acceleration and 3/9 deceleration.
10m
Decant and discard the supernatant.
Resuspend the pellet with the recommended NPB buffer.
Gently rotate the sample in cold room/chiller for 00:10:00 .
Note
Users should optimize lysis timing for different samples. (i.e., 5 minutes for cardiac tissues).
10m
Centrifuge permeabilized nuclei at 900 x g, 4°C, 00:10:00 .
10m
Decant and discard the supernatant.
Resuspend the pellet in 500 uL PBS + RNase In.
Note
For SNARE-seq2: immediately fix nuclei by adding (1:1) 500 uL PBS + Rnase In + 1% paraformaldehyde. (Make 1 mL : 937.5 uL PBS+RI + 62.5 uL 16% methanol-free PFA). Incubate on ice for 00:10:00
QA/QC: Count the nuclei. Mix 1:1 volume of cellular suspension with a staining solution (ie DAPI). Load 10 uL of mixture onto a Biorad Cell Counter slide then count with a BioRad TC20 Cell Counter. Gate 4 uM-6 uM for nuclei sizes.
QA/QC: Check nuclei integrity under fluorescent microscope using DAPI channel. Nuclei should appear distinct, have rounded borders and the majority occurring as singlets.
Note
If high clumping is observed: bring total volume to ~1 mL with PBS+0.1%BSA and filter the sample through a 30 uM CellTrics filter. Pellet 900 x g, 4°C, 00:10:00 . Then resuspend again in 100 uL PBS + Rnase In.
If high debris is observed (low DAPI+): bring volume to ~ 1 mL with PBS+0.1%BSA and pellet: 900 x g, 4°C, 00:10:00 . Then resuspend again in 100 uL PBS + RNase In.