Extraocular muscle stem cells exhibit distinct cellular properties associated with non-muscle molecular signatures

The muscle stem cell (MuSC) population is recognized as functionally heterogeneous. Cranial muscle stem cells, which originate from head mesoderm, can have greater proliferative capacity in culture and higher regenerative potential in transplantation assays when compared to those in the limb. The existence of such functional differences in phenotypic outputs remain unresolved as a comprehensive understanding of the underlying mechanisms is lacking. We addressed this issue using a combination of clonal analysis, live imaging, and scRNA-seq, identifying critical biological features that distinguish extraocular (EOM) and limb (Tibialis anterior, TA) MuSC populations. Time-lapse studies using a MyogenintdTomato reporter showed that the increased proliferation capacity of EOM MuSCs is accompanied by a differentiation delay in vitro. Unexpectedly, in vitro activated EOM MuSCs expressed a large array of distinct extracellular matrix (ECM) components, growth factors, and signaling molecules tha...,

scRNAseq data generation
MuSCs were isolated on BD FACSAriaTM III based on GFP fluorescence and cell viability from Tg:Pax7- nGFP mice (Sambasivan et al., 2009). Quiescent MuSCs were manually counted using a hemocytometer and immediately processed for scRNA-seq. For activated samples, MuSCs were cultured in vitro as described above for four days. Activated MuSCs were subsequently trypsinized and washed in DMEM/F12 2% FBS. Live cells were re-sorted, manually counted using a hemocytometer and processed for scRNA-seq.
Prior to scRNAseq, RNA integrity was assessed using Agilent Bioanalyzer 2100 to validate the isolation protocol (RIN>8 was considered acceptable). 10X Genomics Chromium microfluidic chips were loaded with around 9000 cells and cDNA libraries were generated following manufacturer’s protocol. Concentrations and fragment sizes were determined using Agilent Bioanalyzer and Invitrogen Qubit. cDNA libraries were sequenced using NextSeq 500 and High Output v2.5 (75 cycles) kit..., , # Extraocular muscle stem cells exhibit distinct cellular properties associated with non-muscle molecular signatures

## Description of the data and file structure

The shared object is composed of two distinct folders corresponding to the Quiescent cells and Activated cells. Each folder is organized the same way:

* One folder \"CellRanger_outs\" containing raw and filtered counts matrices as outputed by CellRanger
* One folder \"PREPROCESSED\" containing the Seurat processed R object and a subfolder with the R object used for the scVelo and pyScenic analyses.

Q and ACT refers respectively to Quiescent and Activated cells.

肌肉干细胞（muscle stem cell, MuSC）群体被证实存在功能异质性。起源于头部中胚层的颅部肌肉干细胞，相较于肢体来源的肌肉干细胞，在体外培养中具备更强的增殖能力，且在移植实验中展现出更高的再生潜能。这类功能表型差异的分子机制尚未得到全面阐释，因此其成因至今仍未明确。本研究结合克隆分析、活细胞成像与单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）技术，明确了区分眼外肌（extraocular muscle, EOM）与肢体胫骨前肌（tibialis anterior, TA）肌肉干细胞群体的关键生物学特征。通过Myogenin-tdTomato报告基因开展的延时成像研究显示，眼外肌肌肉干细胞的增殖能力提升，同时伴随体外分化过程的延迟。令人意外的是，体外激活的眼外肌肌肉干细胞可表达大量独特的细胞外基质（extracellular matrix, ECM）成分、生长因子及信号分子……

### 单细胞RNA测序数据生成
研究人员从Tg:Pax7-nGFP小鼠中，基于GFP荧光信号与细胞活力，通过BD FACSAria™ III流式细胞仪分离得到肌肉干细胞（Sambasivan等，2009）。采用血球计数板手动计数静息态肌肉干细胞后，立即进行单细胞RNA测序样本制备。对于激活态样本，肌肉干细胞按前述方法体外培养4天，随后用胰酶消化，以含2%胎牛血清（fetal bovine serum, FBS）的DMEM/F12培养基重悬并洗涤。对活细胞进行二次分选，经血球计数板手动计数后，开展单细胞RNA测序。
在单细胞RNA测序前，使用Agilent Bioanalyzer 2100评估RNA完整性以验证分离流程（RNA完整性指数>8视为合格）。将约9000个细胞上样至10X Genomics Chromium微流控芯片，按照厂商操作规程构建cDNA文库。通过Agilent Bioanalyzer与Invitrogen Qubit测定文库浓度与片段大小，使用NextSeq 500测序仪及High Output v2.5（75循环）试剂盒完成测序……

# 眼外肌肌肉干细胞具有与非肌肉分子特征相关的独特细胞属性

## 数据与文件结构说明
该共享数据集包含两个独立文件夹，分别对应静息态细胞与激活态细胞。两个文件夹的组织格式完全一致：
* 一个名为"CellRanger_outs"的文件夹，内含CellRanger输出的原始计数矩阵与过滤后计数矩阵
* 一个名为"PREPROCESSED"的文件夹，内含Seurat处理后的R对象，以及一个用于scVelo与pyScenic分析的子文件夹及其对应的R对象。
其中Q与ACT分别指代静息态（Quiescent）与激活态（Activated）细胞。