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Sample GSM5059686

Query DataSets for GSM5059686

Status

Public on May 06, 2021

Title

Aged MuSCs 3dpi

Sample type

SRA

Source name

TA and EDL muscle

Organism

Mus musculus

Characteristics

strain: C57BL6J
age: 22-24 months
10x chemistry: v3
timepoint (days post injury): 3

Treatment protocol

C57BL/6 wild-type female mice were obtained from Charles River Breeding Laboratories, the National Institute on Aging, or from a breeding colony at the University of Michigan (UM). 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 were in accordance with the U.S. National Institute of Health (NIH). Young female mice (3-4 months) and aged female mice (20-24 months) were randomly assigned to one of five groups: uninjured, day 3, and day 7 injured (n=4 per group). To induce skeletal muscle injury, mice were first anesthetized with 2% isoflurane and administered a 1.2% barium chloride (BaCl2) solution injected intramuscularly into several points of the tibialis anterior and gastrocnemius muscles for a total of 80 µL per hindlimb.

Growth protocol

n/a

Extracted molecule

total RNA

Extraction protocol

For tissue collection, mice were anesthetized with 3% isoflurane, then euthanized by cervical dislocation, bilateral pneumothorax and removal of the heart. Hind limb muscles (tibialis anterior and gastrocnemius) of control and experimental mice were quickly harvested using sterile surgical tools and placed in separate plastic petri dishes containing cold PBS. Using surgical scissors, muscle tissues were minced and transferred into 50 mL conical tubes containing 20 mL of digest solution (2.5 U/mL Dispase II and 0.2% [~5,500 U/mL] Collagenase Type II in DMEM media per mouse). Samples were incubated on a rocker placed in a 37oC incubator for 60 min with manual pipetting the solution up and down to break up tissue every 30 minutes using an FBS coated 10 mL serological pipette. Once the digestion was completed, 20 mL of F10 media containing 20% heat inactivated FBS was added into each sample to inactivate enzyme activity. The solution was then filtered through a 70 µm cell strainer into a new 50 mL conical tube and centrifuged again at 350xg for 5 min. The pellets were re-suspended in 6 mL of staining media (2% heat inactivated FBS in Hank’s Buffered Salt Solution - HBSS) and divided into separate FACS tubes. The FACS tubes were centrifuged at 350xg for 5 min and supernatants discarded. The cell pellets were then re-suspended in 200 µL of staining media and antibody cocktail containing Sca-1:APC (1:400), CD45:APC (1:400), CD11b:APC (1:400), Ter119:APC (1:400), CD29/b1-integrin:PE (1:200), and CD184/CXCR-4: BIOTIN (1:100) and incubated for 30 minutes on ice in the dark. Cells and antibodies were diluted in 3mL of staining solution, centrifuged at 350xg for 5 min, and supernatants discarded. Pellets were resuspended in 200uL staining solution containing PECy7:STREPTAVIDIN (1:100) and incubated on ice for 20 minutes in the dark. Again, samples were diluted in 3mL staining solution, centrifuged, supernatants discarded, and pellets re-suspended in 200uL staining buffer. Live cells were sorted from the suspension via addition of 1 µg of propidium iodide (PI) stain into each experimental sample and all samples were filtered through 70 μm cell strainers before the FACS. Cell sorting was done using a BD FACSAria III Cell Sorter (BD Biosciences, San Jose, CA) and APC negative, PE/PECy7 double-positive MuSCs were sorted into staining solution for immediate processing.
Freshly isolated MuSCs were sorted into staining solution, enumerated by hemocytometer, and re-suspended into PBS. Cells were loaded into the 10x Genomics chromium single cell controller for each time point and age group 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 NextSeq 500 instrument (Illumina) or NovaSeq instrument (Illumina) with 26 bases for read1 and 98 bases for read2.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

aged_timecourse_cca_filtered_v2.rds

Data processing

Base calling, alignment, filtering, barcode counting, and UMI counting: CellRanger v3.1 (10x Genomics) was used to process raw data. The CellRanger count command was run with default parameters with the exception of the --expect-cells parameter which was set at 10000.
Data filtering and quality control: Datasets were loaded into LIGER using the read10x function followed by createLiger, and cells with less than 300umis were removed. Data scaling, identification of highly variable features, and normalization was performed using LIGER.
Batch correction: Batch effects were removed using LIGER following normalization (k = 30, lambda = 3). Factors dominated by ribosomal or mitochondrial genes were removed. For scVelo analysis, aged timecourse datasets were batch corrected using Seurat v3.
Dimension reduction and community detection: Batch-corrected LIGER objects were imported into Seurat objects after running UMAP dimensional reduction using LIGER. Dimensional reduction and community detection was then performed using RunUMAP (reduction = 'inmf') and FindNeighbors in Seurat.
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: Seurat objects containing raw counts, normalized, scaled, and dimensionally reduced datasets, with metadata for the timepoint, age, and original dataset identity.

Submission date

Feb 02, 2021

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