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| Status |
Public on Nov 01, 2022 |
| Title |
human, Semitendinosus, 01, scRNA-seq |
| Sample type |
SRA |
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| Source name |
Semitendinosus muscle
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| Organism |
Homo sapiens |
| Characteristics |
tissue: Semitendinosus muscle
cell type: Mononuclear cells
developmental stage: young adult
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Semitendinosus muscle from consenting young patients (n = 5; 2 males, 3 females, mean age = 23.4 years) undergoing routine orthopedic surgery for anterior cruciate ligament repair was immediately placed into preservation media (20% FBS in DMEM) following biopsy and transported to the laboratory for immediate single-cell analysis. Upon arrival, muscle sections were washed with PBS, any associated tendons, ligaments, adipose tissue, and blood vessels removed, and tissue weighed. Muscle was then mulched for ~5 mins in a few drops of HBSS using fine surgical scissors. Dissociation media consisting of collagenase D (10 mg/mL, Roche), and 2.4 U/mL dispase II (Sigma) was then added to the samples and the samples were incubated at 37°C for 45 minutes, with periodic agitation. Ten volumes of PBS were added, and the cell-containing solution was filtered through a 70 µm strainer, and centrifuged at 1100 x g for 10 mins. RBCs were lysed and myofiber debris removed using commercially available kits (Qiagen, 158904, Miltenyi Biotec, 130109398, respectively) as per the manufacturer's instructions. Trypan blue viability tests (VWR, K940) were performed on all samples and a minimum of 80% of the cells in each sample were found to be viable. Cells were then fixed in ice-cold methanol for 15 minutes, and stored at -80°C.
Cells were recoved from methanol by centrifugation at 1000 x g for 5 mins at 4°C , and then rehydrated using a wash-resuspension buffer, consisting of 0.04% BSA, 1mM DTT, and 0.2 U/µL RNAse inhibitor in a 3x saline-sodium citrate buffer (in nuclease-free water). All samples were then combined and loaded into the 10x Chromium controller and scRNA-seq performed.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Sequencing of cDNA libraries were performed on Illumina HiSeq 2500 using single-end reads of 91 bp length.
Raw reads were filtered and aligned to GRCh38 human genome assembly, followed by UMI counting and gene expression matrix creation using the cellranger analysis suite (10x Genomics).
Gene expression matrices were analyzed in python 3.7.12 using the scanpy library (1.8.2). Predicted doublets were first removed from the raw data matrix using scrublet. Additionlly, any cells with UMI counts of less than 450 or more than 15000, total genes of less than 250 or greater than 4000, or with greater than 15% mitochondrial genes removed.
The gene expression matrix was first normalized by read depth and then log-transformed, followed by principal component analysis using the top 2000 most variable genes and neighborhood graph construction. Leiden clustering (resolution 0.7) was performed on the neighborhood graph, and clusters were visualized using the uniform manifold approximation and projection method. Significantly differentially expressed genes between clusters were deterined by the Wilcoxon rank sum test and Benjamini-Hochberg correction.
Assembly: GRCh38
Supplementary files format and content: processed_humanSKM.h5ad.gz: processed scanpy object containing clustering result, cluster annotations, and normalized gene expression.
Supplementary files format and content: matrices.tar.gz: gzipped tarball of cellranger output gene expression matrices (raw and filtered) in market exchange (MEX) format.
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| Submission date |
Oct 25, 2022 |
| Last update date |
Nov 02, 2022 |
| Contact name |
Adam Johnston |
| E-mail(s) |
AdamJohnston@dal.ca
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| Organization name |
Dalhousie University
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| Department |
Medical Neuroscience
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| Lab |
Johnston Lab
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| Street address |
1344 Summer Street
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| City |
Halifax |
| State/province |
NS |
| ZIP/Postal code |
B3H0A8 |
| Country |
Canada |
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| Platform ID |
GPL16791 |
| Series (1) |
| GSE216544 |
Identification of underexplored mesenchymal and vascular-related cell populations in human skeletal muscle |
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| Relations |
| BioSample |
SAMN31440538 |
| SRA |
SRX18016387 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6678961_matrices.tar.gz |
51.4 Mb |
(ftp)(http) |
TAR |
| GSM6678961_processed_humanSKM.h5ad.gz |
22.4 Mb |
(ftp)(http) |
H5AD |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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