Isolation of nuclei from frozen human skeletal and cardiac muscle for single nucleus RNA and chromatin assays
Dinh H Diep, Sebastian Preissl, James R Hocker
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
Steps
Section flash-frozen skeletal muscle into aliquots according to desired nuclei yield and store on dry ice or at -80°C .
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.
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.160x g,4°C
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.
900x g,4°C
Decant and discard the supernatant.
Resuspend the pellet with the recommended NPB buffer.
Gently rotate the sample in cold room/chiller for 0h 10m 0s .
Centrifuge permeabilized nuclei at 900x g,4°C .
Decant and discard the supernatant.
Resuspend the pellet in 500 uL PBS + RNase In.
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 .
