Dec 22, 2024

Public workspaceSkeletal muscle extraction from AllProtect Tissue Reagent

DOI
dx.doi.org/10.17504/protocols.io.j8nlk8dp1l5r/v1
  • Elisabeth Heuston1,
  • Ayo P. Doumatey1,
  • Faiza Naz2,
  • Shamima Islam2,
  • Stacie Anderson3,
  • Martha R. Kirby3,
  • Stephen Wincovitch4,
  • Stefania Dell’Orso2,
  • Charles N. Rotimi1,
  • Adebowale Adeyemo1
  • 1Center for Research on Genomics and Global Health, National Human Genome Research Institute, National Institutes of Health;
  • 2Genomic Technology Section, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD;
  • 3NHGRI Flow Cytometry Core, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA;
  • 4Advanced Imaging & Analysis Core, National Human Genome Research Institute, NIH, Bethesda, Maryland, USA
Elisabeth Heuston
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DOI: dx.doi.org/10.17504/protocols.io.j8nlk8dp1l5r/v1
External link: https://www.genome.gov/about-nhgri/Center-for-Research-on-Genomics-and-Global-Health
Protocol Citation: Elisabeth Heuston, Ayo P. Doumatey, Faiza Naz, Shamima Islam, Stacie Anderson, Martha R. Kirby, Stephen Wincovitch, Stefania Dell’Orso, Charles N. Rotimi, Adebowale Adeyemo 2024. Skeletal muscle extraction from AllProtect Tissue Reagent. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk8dp1l5r/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
Created: June 25, 2024
Last Modified: December 22, 2024
Protocol Integer ID: 102401
Keywords: AllProtect Tissue Reagent, Preserved tissue, Skeletal muscle, scRNA-Seq, Single cell RNA sequencing
Funders Acknowledgements:
National Human Genome Research Institute, the National Institute of Diabetes and Digestive and Kidney Diseases, the Center for Information Technology, and the Office of the Director at the National Institutes of Health
Grant ID: 1ZIAHG200362
Disclaimer
Please not that this protocol has been developed specifically for human skeletal muscle tissue preserved in the AllProtect‱ Tissue Reagent and the described equipment configurations. Additional optimization may be necessary for other applications.
Abstract
Single cell genomics analysis often utilizes freshly collected or flash frozen tissue, which is not always available or whose collection is not feasible in research and clinical contexts. Similarly, often the only tissue available on research participants and patients is archived or biobanked. This protocol includes four methods using a combination of flow cytometry and confocal microscopy to extract high quality single cell RNA sequencing data (scRNA-Seq data) from tissues preserved in the nucleic acid stabilizing fixative AllProtect‱ Tissue Reagent.
Image Attribution
Center for Research on Genomics and Global Health Logo.
Logo Credit: Darryl Leja, National Human Genome Research Institute
Materials
List of reagents
ItemSupplier Part number
AllProtect® Tissue ReagentQiagen76405
Phosphate Buffered Saline (PBS) pH 7.2Quality Biological111-056
Bovine Serum AlbuminMillipore SigmaA7888-50G
RNaseOUT™ Recombinant Ribonuclease InhibitorThermoFisher10777019
EASYstrainer, 70 µM, small diameterGreiner Bio-one542170
EASYstrainer, 40 µM, small diameterGreiner Bio-one542140
Nuclei Extraction BufferMiltenyi130-128-024
Nuclei Buffer (20X)10X Genomics2000153
Alexa Fluor® 594 anti-Nuclear Pore Complex Proteins AntibodyBioLegend682202
DAPI solution (1 mg/mL)ThermoFisher62248

Materials

Before start
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  • All solutions must be created fresh the day of the experiment
  • Filter non-detergent buffers with 0.22 um PES strainer
  • Keep all solutions and samples on ice
Tissue homogenization
Tissue homogenization
Thaw tissue stored in AllProtect® Tissue Reagent on iceSampleSample

Serially dilute and exchange AllProtect® Tissue Reagent with PBS until all storage reagent has been removed
Resuspend in 0.5 mL pBSA
Pulverize tissue to single cell suspension with single-use microtube homogenizer
Filter through 70 µm strainer into fresh 1.5 mL tube
Rinse strainer with 0.5 mL pBSA
Centrifuge for 5 min at 300xg, 4C
Remove supernatant and resuspend in 0.5 mL pBSA
Filter through 40 µm strainer into fresh 1.5 mL tube
Rinse strainer with 0.5 mL pBSA
This preparation will subsequently be referred to as filtered cells
Filtered cell (FC) capture preparation
Filtered cell (FC) capture preparation
Start with filtered cells
Remove ~60,000 cells to fresh 1.5 mL tube
Centrifuge for 5 min at 300 xg, 4C
Resuspend 60,000 cells in 30 µL pBSA
Proceed to Chromium Single Cell 5’ Library & Gel Bead Kit v2 sample capture
Filtered nuclei (FN) capture preparation
Filtered nuclei (FN) capture preparation
Start with filtered cells
Remove 250,000 cells to fresh 1.5 mL tube
Centrifuge for 5 min at 300 xg, 4C
Resuspend in 100 µL Nuclei Extraction Buffer
Incubate on ice, 5 min
Centrifuge for 5 min at 300 xg, 4C
Resuspend in 30 µL Nuclei Buffer
Proceed to Chromium Single Cell 5’ Library & Gel Bead Kit v2 sample capture
Staining and flow cytometry enrichment
Staining and flow cytometry enrichment
Start with filtered cells
Remove 50,000 cells as an unstained cell control (bring minimum volume to 100 µL)
Stain 500,000 cells with a final concentration of 25 µg/mL NPC-AF594
Incubate on ice, 30 min
Bring all volumes to 1 mL
Centrifuge for 5 min at 300xg, 4C
Resuspend in 300 µL pBSA
Sort NPC-AF594+ cells on a Becton Dickinson FACS Aria Fusion using 70 µm nozzle into a collection tube on ice
This preparation will subsequently be referred to as sorted cells
Sorted cell (SC) capture preparation
Sorted cell (SC) capture preparation
Start with sorted cells
Remove ~100,000 cells to fresh 1.5 mL tube
Centrifuge for 5 min at 300 xg, 4C
Resuspend 60,000 cells in 30 µL pBSA
Proceed to Chromium Single Cell 5’ Library & Gel Bead Kit v2 sample capture
Sorted nuclei (SN) capture preparation
Sorted nuclei (SN) capture preparation
Start with sorted cells
Remove 250,000 cells to fresh 1.5 mL tube
Centrifuge for 5 min at 300 xg, 4C
Resuspend in 100 µL Nuclei Extraction Buffer
Incubate on ice, 5 min
Centrifuge for 5 min at 300 xg, 4C
Resuspend in 30 µL Nuclei Buffer
Proceed to Chromium Single Cell 5’ Library & Gel Bead Kit v2 sample capture
Staining for confocal microscopy
Staining for confocal microscopy
Start with up to 50,000 sorted cells (sorted cell capture preparation) or 50,000 sorted nuclei (sorted nuclei capture preparation) in 100 µL pBSA
Stain with a final concentration of 100 µg/mL NPC-AF594 + 50 µg/mL DAPI
Incubate on ice, 20 min
Centrifuge for 5 min at 300 xg, 4C
Resuspend in 300 µL pBSA
Transfer suspension to a 35 mm 1.5 coverslip dish
Imaging was performed on an LSM 880 Airyscan Confocal microscope (Zeiss) with a 63x/1.4 oil objective and captured with Zen_2.3 software (Zeiss).