{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"submitter":["Peter Schjerling"],"organism":["Homo sapiens"],"software":["Cell Ranger","Seurat"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-MTAB-15400"],"description":["Mechanical loading promotes structural and functional improvements in muscle and tendon, protecting against injury at their interface – the myotendinous junction (MTJ) and within the tendon matrix. However, the early cellular and molecular mechanisms underlying these adaptations in humans remain poorly understood. To address this, we used single-nucleus RNA sequencing and in situ hybridization to map the acute response of the human muscle-tendon unit to a single bout of eccentric resistance exercise, with a focus on extracellular matrix (ECM) regulation. We identified four transcriptionally distinct fibroblast subtypes with high expression of ECM components COL1A1 and DCN. Three of these subtypes were exercise-responsive: two localized to tendon fascicles or the MTJ, and a third enriched in the interfascicular matrix. Notably, this interfascicular population, marked by PDGFRA, showed the strongest transcriptional response to exercise, including upregulation of PRG4 and VCAN, ECM genes linked to tissue lubrication and resilience. In parallel, we discovered dynamic ECM remodeling in muscle-derived myonuclei, particularly for a distinct subset of type II myonuclei at the MTJ that expanded in number and strongly upregulated COL22A1, a collagen critical for MTJ integrity. Together, these findings reveal a spatially coordinated program of ECM remodeling involving both fibroblasts and myonuclei, highlighting the compartmentalized and cell type-specific nature of early tissue adaptation to mechanical load in the human muscle-tendon unit."],"repository":["biostudies-arrayexpress"],"sample_protocol":["Library Construction - The official Chromium Single Cell 3’ Reagents Kits v3 User Guide (version CG000183 Rev A) was followed for the creation of single nuclei cDNA libraries. Target nuclei recovery was 10000 per sample.","Nucleic Acid Extraction - Snap frozen tendons were cut into smaller pieces (~0.5 mm3) in a -20°C cabinet and ~50 mg  tissue were transferred to 2-ml screw cap tubes containing five 2.3 mm-diameter stainless steel ball bearings (BioSpec Products Inc., Bartlesville, OK). Samples were kept on dry ice to prevent thawing. All further steps were carried out on ice or at 4°C using chilled reagents. After, 0.5 ml Nuclei EZ Lysis Buffer (Sigma) was added to each tube and then homogenized using a FastPrep-24 (MP Biomedicals) at 4.0 m/s for 20 s, cooled down on ice for 5 min, then homogenized once more at 4.0 m/s for 20 s and cooled down on ice for 5 min. The homogenate was transferred to a pre-chilled 2-ml microcentrifuge tube, 0.5 ml chilled Nuclei EZ Lysis Buffer was added to each tube, mixed gently using a wide bore pipette, and incubated on ice for 5 min with two further mixes using a wide bore pipette during the incubation. The homogenate was then filtered through a 70 µm mini strainer (PluriSelect) and centrifuged for 5 min at 500 x g at 4°C. After, the pellet was resuspended in 1.5 ml chilled Nuclei EZ Lysis Buffer by pipetting 10 times and centrifuged for 5 min at 500 x g at 4°C. After, the pellet was equilibrated on ice with 0.5 ml nuclei wash and suspension buffer (2% BSA in PBS (Sigma), 2 mM MgCl2 (Sigma), 0.2 U/µl Protector RNase inhibitor (Roche)) for 5 min. After, 1 ml nuclei wash and suspension buffer was added, the pellet was then resuspended by pipetting 20 times using a 1000 µl pipette tip and then centrifuged for 5 min at 500 x g at 4°C. The pellet was resuspended in 1 ml nuclei wash and suspension buffer by pipetting 20 times using a 1000 µl pipette tip, then filtered through a 40 µm mini strainer (PluriSelect). The filter was washed with 0.5 ml nuclei wash and suspension buffer. For each sample, the suspension was split into two 1.5 ml microcentrifuge tubes containing 20% and 80% of the volume, respectively. The tubes were centrifuged for 5 min at 500 x g at 4°C and the pellets were each resuspended in 200 µl nuclei wash and suspension buffer. Trypan blue (Sigma) and a hemocytometer were used to count the number of nuclei in 5 µl of the suspension containing 20% nuclei. Nuclei suspensions were stored on ice for no more than 30 min before nuclei sorting.  Fluorescence-activated nuclei sorting Nuclei were sorted by fluorescence-activated cell sorting (FACS) to remove non-nuclear materials and any doublets . To the tube containing 80% nuclei suspension, 1.5 µg/ml 7-aminoactinomycin D (7AAD), a fluorescent intercalator that associates with DNA, was added. The tube containing 20% nuclei suspension was used as a negative label control to set up sorting gates. Forward scatter/side scatter gates were used to remove clumps of cells and debris. Single 7AAD-labelled nuclei were sorted into a 5 °C-chilled 1.5 ml microfuge tube coated with nuclei wash and suspension buffer. FACS was performed on a FACSAria III (BD Biosciences). Sorted nuclei suspensions were transported on ice and immediately used for single nuclei cDNA library preparation.","Sample Treatment - A resistance training protocol targeting the hamstring muscles (semitendinosus and gracilis), with an emphasis on slow, eccentric loading, incorporating unilateral and bilateral exercises performed to fatigue under supervised conditions. Patients warmed up for 5 min on an exercise bike and then performed 3 sets of 8 repetitions of the following exercises until exhaustion, unilateral eccentric leg curl (on the exercise leg only) in the prone position to exhaustion, bilateral leg press, bilateral Nordic hamstring leg curl, bilateral leg extension, unilateral eccentric leg curl (on the exercise leg only) in the prone position to exhaustion, and stiff legged lowering of dead lift to exhaustion. Participants rested for two minutes between the different exercises. All exercises were performed under supervision to ensure a proper form and slow tempo was maintained, and all patients were supervised by the same trainer.","Sample Collection - Healthy muscle-tendon tissues (gracilis and semitendinosus) were obtained as waste tissue from ACL reconstruction surgeries. Within 8 minutes of excision, the tissue was immediately placed in ice-cold phosphate-buffered saline (PBS) and kept on ice. To minimize the presence of myonuclei in our dataset, as our focus was on the maintenance of fibrous connective tissue, all visible attached muscle tissue was carefully dissected from the tendon by scraping with a scalpel. This procedure was performed on ice to minimize RNA metabolism. Our pilot experiment confirmed that this method preserved the abundant myonuclei localized to the MTJ. Following dissection, which took ~30 min, tendon tissue was cut into ~50 mg pieces and snap-frozen in individual cryotubes with liquid nitrogen.","Sequencing - Sequencing of the ten libraries (~750M paired reads) was performed using Illumina HiSeq by Genewiz GmbH (Leipzig, Germany)."],"figure_sub":["Organization","MINSEQE Score","Assays and Data","Processed Data","MAGE-TAB Files"],"data_protocol":["Sequence Alignment - Sequencing files were demultiplexed, barcodes processed, mapped to the human genome (Gencode  release 38 (GRCh38.p13) and reads counted using Cell Ranger v7.0.0.","Data Transformation - Count data were log-normalized using Seurat LogNormalize function."],"omics_type":["Unknown","Transcriptomics","Genomics","Proteomics"],"instrument_platform":["10x Chromium","Illumina NovaSeq 6000"],"study_type":["RNA-seq of coding RNA from single cells"],"species":["Homo sapiens"],"pubmed_authors":["Peter Schjerling"],"additional_accession":[]},"is_claimable":false,"name":"Single-nucleus RNA-seq of human tendon and myotendinous junction after acute exercise","description":"Mechanical loading promotes structural and functional improvements in muscle and tendon, protecting against injury at their interface – the myotendinous junction (MTJ) and within the tendon matrix. However, the early cellular and molecular mechanisms underlying these adaptations in humans remain poorly understood. To address this, we used single-nucleus RNA sequencing and in situ hybridization to map the acute response of the human muscle-tendon unit to a single bout of eccentric resistance exercise, with a focus on extracellular matrix (ECM) regulation. We identified four transcriptionally distinct fibroblast subtypes with high expression of ECM components COL1A1 and DCN. Three of these subtypes were exercise-responsive: two localized to tendon fascicles or the MTJ, and a third enriched in the interfascicular matrix. Notably, this interfascicular population, marked by PDGFRA, showed the strongest transcriptional response to exercise, including upregulation of PRG4 and VCAN, ECM genes linked to tissue lubrication and resilience. In parallel, we discovered dynamic ECM remodeling in muscle-derived myonuclei, particularly for a distinct subset of type II myonuclei at the MTJ that expanded in number and strongly upregulated COL22A1, a collagen critical for MTJ integrity. Together, these findings reveal a spatially coordinated program of ECM remodeling involving both fibroblasts and myonuclei, highlighting the compartmentalized and cell type-specific nature of early tissue adaptation to mechanical load in the human muscle-tendon unit.","dates":{"release":"2025-10-10T00:00:00Z","modification":"2025-10-10T09:26:32.586Z","creation":"2025-07-25T11:53:55.744Z"},"accession":"E-MTAB-15400","cross_references":{"EFO":["EFO_0002944","EFO_0004170","EFO_0005684","EFO_0004917","EFO_0005518","EFO_0003816","EFO_0004184","EFO_0003969"]}}