Jun 16, 2025
Isolation of Skeletal Muscle Nuclei from Postmortem fresh frozen Tissue (4 samples)
This protocol is a draft, published without a DOI.
- 1Boston Children's Hospital
Protocol Citation: Pamela Barraza 2025. Isolation of Skeletal Muscle Nuclei from Postmortem fresh frozen Tissue (4 samples). protocols.io https://dx.doi.org/
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: August 15, 2024
Last Modified: June 16, 2025
Protocol Integer ID: 105721
Keywords: isolation of skeletal muscle nuclei, skeletal muscle nuclei, seq for the pediatric cell atlas, postmortem fresh frozen tissue, nuclei extraction protocol, skeletal muscle, pediatric cell atlas, fresh frozen tissue, frozen tissue, nuclei, snrna, postmortem
Funders Acknowledgements:
CZI
Grant ID: DAF2021-237666
Abstract
Nuclei extraction protocol used by Boston Childrens Hospital to produce snRNA-seq for the Pediatric Cell Atlas of Skeletal Muscle.
Protocol materials
Ethanol Pure 200 proof for molecular biologyMerck MilliporeSigma (Sigma-Aldrich)Catalog #E7023-500mL
RNase ZAPGenesee ScientificCatalog #10-228
eppendorf tubes, 5ml
D-Sucrose RNAse freeVWR International (Avantor)Catalog #97061-430
Bleach (Pure Bright Germicial Ultra Bleach)House of Chemicals Inc.Catalog #GG6506
Kimble™ Kontes™ Dounce Tissue Grinders Fisher ScientificCatalog #K885300-0015
Surgical Scissors (114mm or 4.5)VWR International (Avantor)Catalog #82027-578
2M KClInvitrogenCatalog #AM96400
MgCl2Merck MilliporeSigma (Sigma-Aldrich)Catalog #M1028-100mL
SpermineMerck MilliporeSigma (Sigma-Aldrich)Catalog #S3256
EGTA Buffer 0.5M, pH 8.0Fisher ScientificCatalog #40520008
1M Tris-HCl (pH 8.0)Thermo Fisher ScientificCatalog #15568025
Magnesium AcetateMerck MilliporeSigma (Sigma-Aldrich)Catalog #63052-100ML
EDTA (0.5 M), pH 8.0Life TechnologiesCatalog #AM9260G
DTT, 5g (Dithiothreitol)PromegaCatalog #V3151
Nuclease-Free WaterThermo Fisher ScientificCatalog #AM9937
Calcium chloride solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #21115-100ML
50 mL Tube Top Vacuum Filter SystemCorningCatalog #430320
Protector RNase InhibitorMerck MilliporeSigma (Sigma-Aldrich)Catalog #3335402001
PBS without Ca2 or Mg2 Gibco - Thermo Fisher ScientificCatalog #10010-031
BSAMerck MilliporeSigma (Sigma-Aldrich)Catalog #A7409-10ML
Ultracentrifuge TubesBeckmanCatalog #344061
Corning® 100µm Cell StrainerCorningCatalog #431752
Corning® 70µm Cell StrainerCorningCatalog #431751
Chromium Next GEM Chip G Single Cell Kit v410x GenomicsCatalog #1000690
DNA LoBind Tube 1.5ml EppendorfCatalog #022431021
DAPI Solution (1 mg/mL)Thermo FisherCatalog #62248
Douncers and scissors preparation before isolation day
Soak in 10 % (v/v) Bleach (Pure Bright Germicial Ultra Bleach)House of Chemicals Inc.Catalog #GG6506 for 00:20:00
20m
Clean with Ethanol Pure 200 proof for molecular biologyMerck MilliporeSigma (Sigma-Aldrich)Catalog #E7023-500mL
Autoclave Kimble™ Kontes™ Dounce Tissue Grinders Fisher ScientificCatalog #K885300-0015 and 4 Surgical Scissors (114mm or 4.5)VWR International (Avantor)Catalog #82027-578 wrapped in foil
On the day of the experiment: RNase ZAPGenesee ScientificCatalog #10-228 for 00:05:00
5m
Rinse with DI water
Tissue preparation before isolation day
In a cryochamber cut approximately 20 mg of tissue sample and place in 5 mL eppendorf tubes, 5ml on dry ice. Store at -80 °C until isolation day.
Buffers and Preparation (make fresh on day of isolation)
Make 2.5 Molarity (M) D-Sucrose RNAse freeVWR International (Avantor)Catalog #97061-430 solution: 42.8 g in 50 mL water
Prepare the three buffers below
Lysis Buffer
D-Sucrose RNAse freeVWR International (Avantor)Catalog #97061-430
1M Tris-HCl (pH 8.0)Thermo Fisher ScientificCatalog #15568025
Calcium chloride solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #21115-100ML
Magnesium AcetateMerck MilliporeSigma (Sigma-Aldrich)Catalog #63052-100ML
EDTA (0.5 M), pH 8.0Life TechnologiesCatalog #AM9260G
EGTA Buffer 0.5M, pH 8.0Fisher ScientificCatalog #40520008
DTT, 5g (Dithiothreitol)PromegaCatalog #V3151
Nuclease-Free WaterThermo Fisher ScientificCatalog #AM9937
| A | B | C | |
| Reagent | Final [ ] | Volume | |
| 2.5 M Sucrose | 0.32 M | 6.4 mL | |
| 1 M Tris-HCl | 10 mM | 500 uL | |
| 1 M CaCl2 | 5 mM | 250 uL | |
| 1 M MgAcet | 5 mM | 250 uL | |
| 0.5 M EDTA | 2 mM | 200 uL | |
| 0.5 M EGTA | 0.5 mM | 50 uL | |
| 1 M DDT | 1 mM | 50 uL | |
| Water | 42.3 mL | ||
| TOTAL | 50 mL |
Sucrose Buffer
D-Sucrose RNAse freeVWR International (Avantor)Catalog #97061-430
1M Tris-HCl (pH 8.0)Thermo Fisher ScientificCatalog #15568025
Magnesium AcetateMerck MilliporeSigma (Sigma-Aldrich)Catalog #63052-100ML
DTT, 5g (Dithiothreitol)PromegaCatalog #V3151
Nuclease-Free WaterThermo Fisher ScientificCatalog #AM9937
| A | B | C | |
| Reagent | Final [ ] | Volume | |
| 2.5 M sucrose | 2.1 M | 42 mL | |
| 1 M Tris-HCl | 10 mM | 500 uL | |
| 1 M MgAcet | 5 mM | 250 uL | |
| 1 M DTT | 1 mM | 50 uL | |
| Water | 7.2 mL | ||
| TOTAL | 50 mL |
Nuclei Storage Buffer
1M Tris-HCl (pH 8.0)Thermo Fisher ScientificCatalog #15568025
2M KClInvitrogenCatalog #AM96400
MgCl2Merck MilliporeSigma (Sigma-Aldrich)Catalog #M1028-100mL
SpermineMerck MilliporeSigma (Sigma-Aldrich)Catalog #S3256
Nuclease-Free WaterThermo Fisher ScientificCatalog #AM9937
| A | B | C | |
| Final concentration | Final [ ] | Volume | |
| 1 M Tris-HCl | 10 mM | 125 uL | |
| 1 M KCl | 70 mM | 850 uL | |
| 1 M MgCl2 | 10 mM | 125 uL | |
| 1 M Spermine | 1.5 mM | 18.7 uL | |
| Water | 11.356 mL |
Filter-sterilize buffers through 0.22 µm 50 mL Tube Top Vacuum Filter SystemCorningCatalog #430320 into the provided 50 mL conical tube. The same filter can be used for the lysis and the sucrose buffer. Use a separate filter for the nuclei storage buffer.
Transfer 25 mL of lysis buffer into a clean tube and add 125 µL of RNase inhibitors, transfer
20 mL of sucrose buffer into a new tube and add 100 µL of Protector RNase InhibitorMerck MilliporeSigma (Sigma-Aldrich)Catalog #3335402001 , and
transfer 5 mL of nuclei storage buffer into a new tube and add 25 µL of Protector RNase InhibitorMerck MilliporeSigma (Sigma-Aldrich)Catalog #3335402001 .
Make 40 mL of 1 % (v/v) BSAMerck MilliporeSigma (Sigma-Aldrich)Catalog #A7409-10ML in PBS without Ca2 or Mg2 Gibco - Thermo Fisher ScientificCatalog #10010-031 (1.140 mL of premade 35 % (v/v) BSA to 38.860 mL 1X PBS)
Add 10 mL of 1 % (v/v) BSAMerck MilliporeSigma (Sigma-Aldrich)Catalog #A7409-10ML to 4 Ultracentrifuge TubesBeckmanCatalog #344061 On ice
Tissue homogenization
1h 45m
Transfer tissue from dry ice to wet ice
Add 1 mL of lysis buffer with Protector RNase InhibitorMerck MilliporeSigma (Sigma-Aldrich)Catalog #3335402001 to tissue
Use Surgical Scissors (114mm or 4.5)VWR International (Avantor)Catalog #82027-578 to cut tissue while On ice until tissue can be pipetted out
Add tissue to Kimble™ Kontes™ Dounce Tissue Grinders Fisher ScientificCatalog #K885300-0015 homogenizers
Add 3 mL lysis buffer to the 5 mL eppendorf tubes, 5ml to rinse and then transfer to Kimble™ Kontes™ Dounce Tissue Grinders Fisher ScientificCatalog #K885300-0015 homogenizers
Homogenize (~30X, slowly) using A part douncer
Transfer small volume to glass slide and check under the microscope to see that the cells are fragmented
Filter the entirety of the volume through a Corning® 100µm Cell StrainerCorningCatalog #431752 into a new tube
Filter the entirety of the volume through a Corning® 70µm Cell StrainerCorningCatalog #431751 into a new tube
Centrifuge tubes at 700 x g, 4°C for 00:10:00
10m
During the spin, remove 1 % (v/v) BSAMerck MilliporeSigma (Sigma-Aldrich)Catalog #A7409-10ML from Ultracentrifuge TubesBeckmanCatalog #344061 and then add 5 mL sucrose buffer with Protector RNase InhibitorMerck MilliporeSigma (Sigma-Aldrich)Catalog #3335402001 to the coated tubes
Take the spun nuclei and remove the supernatant by pipetting it out into another 50 mL tube so as not to disturb the pellet and to save it if you lose it
Resuspend the nuclei with 1 mL lysis buffer and pipette gently 10X
Add that 1 mL gently to the top of the sucrose buffer in the Ultracentrifuge TubesBeckmanCatalog #344061 (lysis should float and create a visible gradient)
Weight and balance Ultracentrifuge TubesBeckmanCatalog #344061 using lysis buffer with Protector RNase InhibitorMerck MilliporeSigma (Sigma-Aldrich)Catalog #3335402001
Spin the nuclei at 600 x g, 4°C, 01:00:00
1h
During the spin, start 10X protocol Chromium Next GEM Chip G Single Cell Kit v410x GenomicsCatalog #1000690 (page 30) by reconstituting plasmid (00:30:00 ) and making master mix without RT Enzyme E and adding 3 µL of Protector RNase InhibitorMerck MilliporeSigma (Sigma-Aldrich)Catalog #3335402001 per sample
30m
Pipette 24.4 µL of master mix into DNA LoBind Tube 1.5ml EppendorfCatalog #022431021 and label accordingly
After spin, discard the supernatant by pipetting out supernatant and being very careful not to dislodge the pellet
Dissolve nuclei pellet in 1 mL nuclei storage buffer with Protector RNase InhibitorMerck MilliporeSigma (Sigma-Aldrich)Catalog #3335402001 - if the pellet is very small, resuspend in 500 µL
Filter the 1 mL through the 20 µm FACS tubes (gently tap tubes until solution goes through)
Add DAPI Solution (1 mg/mL)Thermo FisherCatalog #62248 at 1:1000 dilution and incubate for 00:05:00
5m
FACS sort into master mix with Protector RNase InhibitorMerck MilliporeSigma (Sigma-Aldrich)Catalog #3335402001
Add 7 µL RT Enzyme E into each sample tube and measure. Using the same tip, pipette the total final volume (do not discard tip)
Calculate the amount of 1X PBS without Ca2 or Mg2 Gibco - Thermo Fisher ScientificCatalog #10010-031 needed to bring total volume to 60 µL and add with a different tip
Use previous tip to pipette mix cell suspension 5X and proceed with 10X protocol loading