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Sample GSM4977802

Query DataSets for GSM4977802

Status

Public on Apr 04, 2022

Title

VML_D7_3mm_F

Sample type

SRA

Source name

Quadriceps muscle

Organism

Mus musculus

Characteristics

strain: C57BL6J
tissue: Quadriceps muscle
defect: 3mm
gender: female
timepoint (days post injury): 7

Treatment protocol

C57BL/6 wild-type female and male mice were obtained from Jackson Laboratories. All mice were fed normal chow ad libitum and housed on a 12:12 hour light-dark cycle under UM veterinary staff supervision. All procedures were approved by the University Committee on the Use and Care of Animals at UM and the Institutional Animal Care and Committee and were in accordance with the U.S. National Institute of Health (NIH). Young mice (3-4 months) were first anesthetized with 1.5% isoflurane and administered 10mg/kg buprenorphine. Hair was removed from the hindlimbs using clippers, followed by Nair hair removal cream. The surgical area was wiped with proprium iodide followed by 70% ethanol three times to sterilize. A 1cm incision was made in the skin on the anterior side of each quadricep, followed by the removal of a 2mm or 3mm full depth muscle section from the medial quadricep from the distal half of the muscle. A suture was placed in the muscle to identify the location of the injury, and the skin was sutured closed. Dermal sutures were removed 7 days post-surgery.

Extracted molecule

total RNA

Extraction protocol

Mouse quadriceps were extracted, and separately weighed using sterile surgical tools and placed into separate petri dishes containing ice-cold PBS. Using surgical scissors, muscle tissues were minced and collagenase type II (Gibco 17101015, 0.2%) and dispase II (Sigma D4693, 2.5U/mL) were added to 10mL of DMEM (Gibco-Invitrogen Cat#10569010) per mouse. Samples were placed on rocker in a 37˚C incubator for 1 hour and mixed by pipette every 30 minutes. The enzymes within the slurry were then inactivated by addition of 20% heat-inactivated fetal bovine serum (HI-FBS) in Ham’s F10 media (Gibco-Invitrogen # 11550-043). The solution was passed through a 70um cell strainers, centrifuged, washed, and counted. Cells were then divided for fluorescent activated cell sorting (FACS) enrichment of viable cells and removal of debris
Freshly isolated cells were briefly stained with Propidium Iodide (PI)and FACS sorted to remove dead cells (PI positive) and debris. 10,000-16,000 cells were loaded into the 10x Genomics chromium single cell controller and single cells were captured into nanoliter-scale gel bead-in-emulsions (GEMs). cDNAs were prepared using the single cell 3′ Protocol as per manufacturer’s instructions and sequenced on a NovaSeq 6000 flow cell (Illumina) with 26 bases for read1 and 98x8 bases for read2.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Data processing

Base calling, alignment, filtering, barcode counting, and UMI counting: 10x CellRanger v2.0.0 software's mkfastq and count commands were run with default parameters except expect-cells=10000. Alignment was performed to the mm10 reference genome.
Data filtering and quality control: Matrix files were imported into R using the Seurat package. Genes expressed in less than 200 cells or cells expressing less than 3 genes were removed. Seurat objects were merged and cells with >10% mitochondrial gehes or over 75000 reads were deemed low quality and removed. Libraries were normalized and scaled using default parameters.
Batch correction: Technical batch effects were regressed out using Seurat v3s FindIntegrationAnchors and Integrate data functions.
Dimension reduction and community detection: Dimensional reduction was performed in Seurat using UMAP followed by community detection using the louvain algorithm with a resolution of 0.2. Cluster markergenes were determined in Seurat using the Wilcoxon sum-rank test.
Celltype annotation: Cell types were annotated using scCATCH to compare with mouse muscle databases in combination with markergene expression.
Genome_build: mm10
Supplementary_files_format_and_content: Large Seurat object containing merged, processed matrix file as well as raw counts, scaled data, variable features, metadata (condition, timepoint, defect size, mitochondrial gene fraction, seurat clusters, celltypes), and reductions (iNMF and umap).

Submission date

Dec 16, 2020

Last update date

Feb 01, 2023

Contact name

Carlos Andres Aguilar

E-mail(s)

caguilar@umich.edu

Phone

734-764-8557

Organization name

University of Michigan