biostudies-arrayexpressMengyuan ZhangRattus norvegicushttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-16550This study presents a cross-species single-cell transcriptomic atlas of the meniscus. The analysis includes genetic lineage tracing mice, wild-type rats, and dogs. Genetic Lineage Tracing Mice: ACTA2-CreERT mice (generated via CRISPR/Cas9 knock-in at the Acta2 locus) were crossed with R26-CAG-LSL-tdTomato reporter mice. All mice received tamoxifen (75 mg/kg, i.p. for 5 days) to induce tdTomato expression in ACTA2-lineage cells. Menisci were harvested from an early post-induction cohort at 2 weeks (n=13) and a late cohort at 10 weeks of age (n=10). Other Species: Menisci were also analyzed from 12-week-old male Sprague-Dawley rats (n=4) and 12-month-old male beagles (n=2). Single-cell suspensions were captured and barcoded using the BD Rhapsody system. Libraries were constructed with the BD Resolve Whole-Transcriptome Amplification workflow and sequenced on Illumina platforms. Bioinformatic analysis was performed using Seurat for integration, clustering, and annotation of cell types, followed by subpopulation analysis and trajectory inference.biostudies-arrayexpressSample Treatment - Tamoxifen induction: ACTA2-CreERT; R26-tdTomato mice received intraperitoneal injections of tamoxifen (75 mg/kg body weight) dissolved in corn oil, once daily for five consecutive days.Sample Collection - Meniscal tissues were collected from: 1) Genetic Lineage Tracing Mice: ACTA2-CreERT; R26-tdTomato mice (tamoxifen-induced, 75 mg/kg, i.p. for 5 days). Menisci were harvested at 2 weeks (n=13) and 10 weeks of age (n=10). 2) Other Species: Menisci from 12-week-old male Sprague-Dawley rats (n=4) and 12-month-old male beagles (n=2). All tissues were placed in cold PBS post-dissection for immediate processing.Library Construction - Libraries were constructed using the BD Rhapsody system. Single cells and barcoded beads were co-encapsulated in microwells. Poly-adenylated RNA was captured, reverse-transcribed, and each cDNA was tagged with a cell label and UMI. Amplification used the BD Resolve Whole-Transcriptome Amplification kit (second-strand synthesis, adaptor ligation, PCR). Final libraries were prepared by random priming PCR to enrich 3‘ end fragments.Nucleic Acid Extraction - Single-cell suspensions were prepared by enzymatic digestion (e.g., collagenase) of minced meniscal tissue. The suspension was filtered, washed, and resuspended. Cell viability and concentration were assessed via trypan blue exclusion and counting. This protocol yields intact single cells; no bulk nucleic acid extraction was performed.Sequencing - The final single-cell cDNA libraries were sequenced on Illumina platforms (NovaSeq 6000, project-dependent) following the standard manufacturer's sequencing protocol. The sequencing was typically performed in a paired-end mode to simultaneously read the transcript-derived sequence and the cell/UMI barcode. Base calling and demultiplexing were performed using Illumina's proprietary software.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - Gene expression matrices were generated by counting unique UMIs per gene per cell. Data normalization and transformation were performed using the Seurat package (v5.1.0) in R. The steps included: 1. Quality Control: Cells with low/high UMI counts or high mitochondrial gene percentage were filtered. 2. Normalization: Gene expression counts for each cell were normalized by the total counts for that cell, multiplied by a scale factor (10,000), and log-transformed using the LogNormalize method. 3. Feature Selection: Highly variable genes were identified for downstream analysis. 4. Integration & Scaling: For cross-species analysis, gene symbols were standardized. Datasets were integrated using Canonical Correlation Analysis (CCA), and expression values were scaled and centered.UnknownTranscriptomicsGenomicsProteomicsBiological safety cabinet, Centrifuge, Cell strainer, Automated cell counter/HemocytometerSurgical dissection toolsBD Rhapsody systemComputational server/cluster, Software: R (v4.3.x), Seurat package (v5.1.0)Illumina NovaSeq XMicrosyringe for intraperitoneal injectionRNA-seq of coding RNA from single cellsRattus norvegicusMengyuan ZhangfalseSingle-cell RNA sequencing of meniscus from ACTA2-lineage traced mice, rats and dogsThis study presents a cross-species single-cell transcriptomic atlas of the meniscus. The analysis includes genetic lineage tracing mice, wild-type rats, and dogs. Genetic Lineage Tracing Mice: ACTA2-CreERT mice (generated via CRISPR/Cas9 knock-in at the Acta2 locus) were crossed with R26-CAG-LSL-tdTomato reporter mice. All mice received tamoxifen (75 mg/kg, i.p. for 5 days) to induce tdTomato expression in ACTA2-lineage cells. Menisci were harvested from an early post-induction cohort at 2 weeks (n=13) and a late cohort at 10 weeks of age (n=10). Other Species: Menisci were also analyzed from 12-week-old male Sprague-Dawley rats (n=4) and 12-month-old male beagles (n=2). Single-cell suspensions were captured and barcoded using the BD Rhapsody system. Libraries were constructed with the BD Resolve Whole-Transcriptome Amplification workflow and sequenced on Illumina platforms. Bioinformatic analysis was performed using Seurat for integration, clustering, and annotation of cell types, followed by subpopulation analysis and trajectory inference.2026-04-30T00:00:00Z2026-04-30T01:01:52.138Z2026-01-21T14:29:01.505ZE-MTAB-16550ERP187983EFO_0002944EFO_0004170EFO_0005684EFO_0005518EFO_0003816EFO_0004184EFO_0003969