Summary: Skeletal muscle is a complex heterogeneous tissue comprised of diverse muscle fiber and non-fiber cell types that, in addition to movement, influences other systems such as immunity, metabolism and cognition. We investigated gene expression patterns of resident human skeletal muscle cells using single-cell RNA-seq of dissections from vastus lateralis. We generate transcriptome profiles of 11 mononuclear human skeletal muscle mononuclear cell types, including immune, endothelial, pericyte and satellite cells. We delineate two fibro-adipogenic progenitor cell subtypes that may contribute to heterotopic ossification and muscular dystrophy fibrosis under pathological conditions. An important application of cell type signatures is for computational deconvolution of cell type specific changes using data from bulk transcriptome experiments. Analysis of transcriptome data from a 12 week resistance training study using the human skeletal muscle cell-type signatures revealed significant changes in specific mononuclear cell-type proportions related to age, sex, acute exercise and training. This characterization of human skeletal muscle cell subtypes will resolve cell type specific changes in large-scale physical activity muscle transcriptome studies and can further the understanding of the diverse effects of exercise and the pathophysiology of muscle disease.
Overall Design: Single cell RNA sequencing was conducted on four samples of mononuclear muscle cells isolated from a single vastus lateralis biopsy.
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Growth Protocol: | - |
Treatment Protocol: | - |
Extract Protocol: | Four individual samples of dissociated cryopreserved cells (non-fiber cells only) obtained from a single human muscle biopsy were thawed based on the 10X Genomics Demonstrated Protocol (Manual Part Number CG00039 Rev C). Briefly, cryovials were removed from liquid nitrogen storage and immediately thawed in a 37℃ waterbath for 2 minutes. 1ml of the thawed cells were transferred into individual tubes, the cryovials were rinsed with 1ml of warm complete growth medium and the rinse medium was added dropwise (1 drop per 5 sec) to each tube containing the thawed cells. The cells were serially diluted with complete growth medium a total of 3 times by 1:1 volume dropwise additions with 1 minute wait before each addition. Next the cells were centrifuged at 300 rcf for 5 minutes and resuspended in complete growth medium. Cells were filtered through a 40μm cell strainer, counted, and the volume of medium was adjusted for the final concentration to be 1,000 cells/μl |
Library Construction Protocol: | Single-cell encapsulation with beads using the 10X Genomics Chromium Single Cell 3' v2 kit (PN 120235 and PN 120234 Module 1) was performed following manufacturer instructions (User Guide Rev A, 10X Genomics, Pleasanton, CA, USA). Briefly, 4 wells of a 10X microfluidic chip (PN 120236) were loaded each with an individual sample to target 5,000 cells per sample. Single-cell gel beads in emulsion (GEMs) were generated and reverse-transcription was performed in the emulsion prior to 12 cycles of cDNA amplification. Quality control and quantification of the amplified cDNA were assessed by Bioanalyzer. Libraries were constructed according to manufacturer instructions (PN 120234 Module 2). Each library was tagged with a different index for multiplexing (PN 120262) during sequencing. Library quality controls were assessed by Bioanalyzer and quantified by Qubit and quantitative PCR (KAPA Biosystems Quantification for Illumina platforms). Sequencing was performed on an HiSeq 2500 (Illumina, Inc., San Diego, CA) using the following read length: 26 bp Read1 (cell barcode(first 16 nt) + UMI(next 10 nt)), 8 bp i7 index (sample index), and 98 bp Read2 (insert). |
Molecule Type: | poly(A)+ RNA |
Library Source: | |
Library Layout: | PAIRED |
Library Strand: | Forward |
Platform: | ILLUMINA |
Instrument Model: | Illumina HiSeq 2500 |
Strand-Specific: | Specific |
Data Resource | GEN Sample ID | GEN Dataset ID | Project ID | BioProject ID | Sample ID | Sample Name | BioSample ID | Sample Accession | Experiment Accession | Release Date | Submission Date | Update Date | Species | Race | Ethnicity | Age | Age Unit | Gender | Source Name | Tissue | Cell Type | Cell Subtype | Cell Line | Disease | Disease State | Development Stage | Mutation | Phenotype | Case Detail | Control Detail | Growth Protocol | Treatment Protocol | Extract Protocol | Library Construction Protocol | Molecule Type | Library Layout | Strand-Specific | Library Strand | Spike-In | Strategy | Platform | Instrument Model | Cell Number | Reads Number | Gbases | AvgSpotLen1 | AvgSpotLen2 | Uniq Mapping Rate | Multiple Mapping Rate | Coverage Rate |
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