HFD post-CTX day 7
Mus musculus
Protocols: Mononuclear cells from the regenerating limb muscles of 4 days or 7days after cardiotoxin (CTX) injection or uninjured muscles were prepared as follows. CTX-injected or uninjured hind limb muscles were carefully dissected to remove attached tendons, nerves, blood vessels, and fat tissue. Trimmed muscles were minced and digested with collagenase II (2mg/ml, Worthington Biochemical Corporation, New Jersey, USA) and Dnase Ⅰ (20ug/ml) in Hanks' Balanced Salt Solution (HBSS) (+) without phenol red (Wako Pure Chemical Industries) for 60 min at 37 °C. Digested muscles were passed through an 18-gauge needle several times and further digested for 30 min at 37 °C. Muscle slurries were filtered through a 100 μm cell strainer (BD Falcon) and through a 40 μm cell strainer (BD). Erythrocytes were eliminated by treating the cells with RBC lysis buffer (Biolegend). Mononuclear cells isolated from muscles were stained with anti-CD31 (Biolegend), anti-CD45 (Biolegend), and anti- SMC2.6(a gift from So-ichiro Fukada., Osaka University, Osaka, Japan) antibodies to exclude endothelial cells, immune cells, satellite cells, respectively, and anti-PDGFRα (R&D) antibodies for collection. Primary antibodies and secondary reagents used for cell staining are listed in the Key resource table. Cell sorting was performed using a Sony SH800 cell sorter (Sony). Debris and dead cells were excluded by forward scatter, side scatter and Propidium Iodide (PI, Biolegend) gating. The analysis of stained cells was performed by Sony SH800 cell sorter and/or Attune NxT (Thermo Fisher) analyzer, and data were processed by FlowJo Version 10 software (FlowJo). To create the acute muscle injury (AMI) model, 100µL of cardiotoxin CTX (10uM in saline, Sigma-Aldrich) was injected into the tibialis anterior (TA) muscle of mice with 29 gauge needle under anesthesia. After PDGFRα-positive cells were sorted from murine muscles as mentioned, and single-cell suspensions were washed and resuspended in PBS at a concentration of at least 1000cells/uL. Single-cell RNA-sequencing libraries were then prepared using the Chromium Single Cell 30 reagent kit v2 (10X Genomics) in accordance with the manufacturer's protocol.
ENA-FIRST-PUBLIC
2021-01-15T04:14:56Z
ENA-LAST-UPDATE
2020-11-09T12:33:04Z
External Id
SAMEA7540786
INSDC center name
Department of Pathophysiology, Graduate School of Medicine, Osaka City University
INSDC first public
2021-01-15T04:14:56Z
INSDC last update
2020-11-09T12:33:04Z
INSDC status
public
Submitter Id
E-MTAB-9715:HFD post-CTX day 7
age
20
broker name
ArrayExpress
cell type
mononuclear cell
common name
house mouse
diet
high fat diet
genotype
wild type genotype
inferred cell type - authors labels
fibroadipogenic progenitor
organism part
hind limb muscle
sample name
E-MTAB-9715:HFD post-CTX day 7
scientific_name
Mus musculus
sex
female
strain
C57BL/6