organism: Mus musculus
description: 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.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2021-01-15T04:14:56Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2020-11-09T12:33:04Z
attributes.tag: External Id
attributes.value: SAMEA7540788
attributes.tag: INSDC center name
attributes.value: Department of Pathophysiology, Graduate School of Medicine, Osaka City University
attributes.tag: INSDC first public
attributes.value: 2021-01-15T04:14:56Z
attributes.tag: INSDC last update
attributes.value: 2020-11-09T12:33:04Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-9715:ND post-CTX day 4
attributes.tag: age
attributes.value: 20
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell type
attributes.value: mononuclear cell
attributes.tag: common name
attributes.value: house mouse
attributes.tag: diet
attributes.value: normal diet
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: inferred cell type - authors labels
attributes.value: fibroadipogenic progenitor
attributes.tag: organism part
attributes.value: hind limb muscle
attributes.tag: sample name
attributes.value: E-MTAB-9715:ND post-CTX day 4
attributes.tag: scientific_name
attributes.value: Mus musculus
attributes.tag: sex
attributes.value: female
attributes.tag: strain
attributes.value: C57BL/6