Oct 29, 2025
JMN-MSMP Mouse Muscle Fibrosis scRNA-seq
- Lexi Rindone1
- 1Johns Hopkins University
Protocol Citation: Lexi Rindone 2025. JMN-MSMP Mouse Muscle Fibrosis scRNA-seq. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg31n4zl25/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
We use this protocol and it's working
Created: October 28, 2025
Last Modified: October 29, 2025
Protocol Integer ID: 231020
Keywords: seq dataset of murine muscle fibrosi, murine muscle fibrosi, muscle fibrosi, enriched scrna, profiling of senotype, polycaprolactone, seq dataset, volumetric muscle loss injury, jmn
Funders Acknowledgements:
NIH
Grant ID: 1U54AG079779
Abstract
This protocol was used to generate a CD45+ cell-enriched scRNA-seq dataset of murine muscle fibrosis induced via a volumetric muscle loss injury and synthetic biomaterial (polycaprolactone) implant. This tissue is enriched in p16+ and p21+ cells, enabling the profiling of senotypes involved in muscle fibrosis.
Troubleshooting
Tissue Harvest and Cell Isolation
Euthanize mice according to approved IACUC protocol.
Harvest each quadricep from each animal. Take extra care to avoid cutting the femoral artery, as excess bleeding will contaminate the tissue with immune cells from the blood.
Place both quads in a petri dish containing chilled RPMI-1640 with L-Glutamine + 25 mM HEPES (Base Media). Keep petri dishes on ice.
Dice quads into <3 mm pieces using a razor blade.
Add 1:1 v/v of pre-warmed solution of Base Media + 1 mg/mL Liberase TL + 0.4 mg/mL DNAse I (2X Digestion Media) to yield a 1X digestion solution.
Incubate quads at 37°C with gentle agitation for 45 min.
Add 2:1 v/v of chilled Base Media + 1% BSA to neutralize the Digestion Media.
Strain tissues through a 100 µm cell strainer into a 50 mL tube. Use the end of a syringe plunger to force cells through the strainer. Rinse the strainer with 1X DBPS twice, mashing the tissues in between each wash.
Centrifuge cells at 400 g for 5 min.
Resuspend cells in 5 mL of ACK Lysis Buffer at room temperature for 3 min.
Add >20 mL of 1X DBPS and strain cells through a 40 µm strainer.
Centrifuge cells at 400 g for 5 min.
Dead Cell Magnetic Bead Removal
Resuspend cells in Dead Cell Removal MicroBeads (Miltenyi # 130090101; 10^7 cells/100 µL) for 15 minutes at room temperature.
Prepare LS column with 3 mL of 1X Binding Buffer.
Add 3 mL of 1X Binding Buffer to cells and add to LS column to enable flow-through of live cells.
Collect the live cells from the flow-through solution.
Wash column with 4 x 3 mL of 1X Binding Buffer.
CD45+ Cell Enrichment
Resuspend cells in 80 µL of Wash Buffer (1X DBPS + 1% w/v BSA + 1 mM EDTA) per 10^7 cells.
Add 20 µL of CD45 Microbeads (Miltenyi # 130045801) per 10^7 cells (yielding 100 µL of solution per 10^7 cells) and incubate for 15 minutes at 4°C.
Wash cells by adding 2 mL of Wash Buffer per 10^7 cells.
Spin down cells at 300 g for 10 minutes.
Resuspend cells in 500 µL of Wash Buffer for 10^8 cells.
Prime column 3 x 3 mL of Wash Buffer.
Add cell suspension to column.
Wash column 3 x 3 mL of Wash Buffer.
Collect flow-through cell solution. This solution contains the CD45- cell fraction.
Remove column from magnet, add 5 mL of Wash Buffer to column, and plunge over a new tube. Collect flow-through cell solution. This is the CD45+ cell fraction.
Count cells in the CD45+ and CD45- cell fractions.
Combine cell solutions to yield a 1:1 mixture of CD45+ and CD45- cells.
Submit samples to sequencing core to perform 10X 3’ Chromium scRNA-seq. (We use the Johns Hopkins Single Cell & Transcriptomics Core.)
Single Cell Sequencing
Prepare libraries using 10X 3’ Chromium v3.1 chemistry protocols and reagents.
Sequence libraries using NovaSeq platform (Illumina) with S2 100 flow cell at a target depth of 100,000 unique reads/cell.