| Experiment Id | GSE223751 | Name | Single-cell RNA sequencing reveals cellular and molecular heterogeneity in fibrocartilaginous enthesis formation |
| Experiment Type | RNA-Seq | Study Type | Baseline |
| Source | GEO | Curation Date | 2024-02-15 |
| description | The attachment site of the rotator cuff (RC) is a classic fibrocartilaginous enthesis, which is the junction between bone and tendon with typical characteristics of a fibrocartilage transition zone. Enthesis development has historically been studied with lineage tracing of individual genes selected a priori, which does not allow for the determination of single-cell landscapes yielding mature cell types and tissues. Here, in together with open source GSE182997 datasets (3 sample) provided by Fang et al, we applied Single-cell RNA sequencing (scRNA-seq) to delineate the comprehensive postnatal RC enthesis growth and the temporal atlas from as early as postnatal day 1 up to postnatal week 8. And we furtherly performed single cell spatial transcriptomic sequencing on postnatal day 1 mice enthesis, in order to deconvoluted bone-tendon junction (BTJ) chondrocytes onto spatial spots. In summary, we deciphered the cellular heterogeneity and the molecular dynamics during fibrocartilage differentiation. Combined with current spatial transcriptomic data, our results provide a transcriptional resource that will support future investigations of enthesis development at the mechanistic level and may shed light on the strategies for enhanced RC healing outcomes. The humeral head- supraspinatus tendon samples were dissected from the left shoulders of C57/BL6 mice at postnatal day-1, day-7, day-14, and day-28. In general, samples were harvested from pooled sibling limbs of two litters (five to six limbs per pool). Following dissection, the humeral heads and tendons were trimmed to retain the enthesis part, and all the samples were minced immediately and digested in type I collagenase (1mg/ml, Gibico) and type II collagenase (1mg/ml, Gibico) diluted in low-glucose DMEM (Gibco) solution at 37 °C for 30-40 min. Freshly isolated cells were resuspended into FACS buffer containing 2% FBS (Gbico) in PBS. Cell suspensions were stained with antibodies including Ter119-Alexa700 and Cd45- Alexa700 (Biolengend) to remove blood cells. DAPI (BD) stain was used to exclude dead cells. Flow cytometry was performed on BD FACS Aria II, single cells were gated using doublet-discrimination parameters and collected in FACS buffer. 8000-10,000 cells were loaded for each age group by Chromium instrument and its chemistry kit V3 (10X Genomics) according to the manufacturer's guidance. Each cell was encapsulated with a barcoded Gel Bead in a single partition, then amplified to generate single-cell cDNA libraries and sequenced on an Illumina NovaSeq 6000 platform at a sequencing depth of ~500 million reads. The Cellranger pipeline (version 6.1.1) was used to align the raw reads to the mouse reference genome GRCm38 and to generate feature-barcode matrices. All the low-quality reads were filtered with default parameters. |
