RNA-sequencing of soleus muscle from Mustn1 heterozygous KO-first mice and wildtype littermates

To uncover novel genes and molecular pathways important for skeletal muscle remodeling and regeneration, we used a mouse hindlimb unloading and reloading protocol. We then performed analysis of global gene expression by RNA-sequencing (RNA-seq), looking at the transition from muscle unloading to reloading (GSE237099). This study focuses on the microprotein Mustn1 (Musculoskeletal embryonic nuclear protein 1, also known as Mustang), which consists of 82 amino acids. We performed RNA-sequencing of soleus muscle from 8-week-old mice carrying one allele (heterozygous) of Mustn1 tm1a (KO-first) and wildtype littermates. RNA-sequencing analysis of soleus muscles from Mustn1 KO-First Heterozygous (HET) mice and wild-type (WT) littermate controls at 8 weeks of age. Total RNA was extracted using TRI reagent (Sigma-Aldrich ref. T9424) according to the manufacturer's instructions. RNA was further purified using a Direct-zol RNA Miniprep kit (Zymo Research) including on-column DNAse I treatment. RNA quality was assessed using a 2100 Bioanalyzer system (Agilent Technologies) with an RNA integrity number (RIN) greater than 8 as a threshold. Samples were purified by poly-A enrichment before library preparation using Illumina TruSeq Stranded mRNA and sequenced on Illumina HiSeq2500 (single reads, 50 bp, 30M reads per sample).为揭示调控骨骼肌重塑与再生的新型基因及分子通路，我们采用小鼠后肢去负荷-再加载造模方案，通过RNA测序（RNA-sequencing, RNA-seq）开展全基因组基因表达分析，聚焦肌肉从去负荷向再加载的转变过程（数据集编号GSE237099）。本研究聚焦微蛋白Mustn1（Musculoskeletal embryonic nuclear protein 1，亦称Mustang），该蛋白由82个氨基酸残基组成。我们对8周龄的Mustn1 tm1a（KO-first）杂合子（heterozygous, HET）小鼠及其野生型（wild-type, WT）同窝对照仔鼠的比目鱼肌开展RNA测序分析。总RNA采用TRI试剂（Sigma-Aldrich，货号T9424）严格依照厂商说明书提取，随后使用Direct-zol RNA Miniprep试剂盒（Zymo Research）完成进一步纯化，并同步进行柱上DNA酶I消化处理。采用2100 Bioanalyzer系统（Agilent Technologies，安捷伦科技）评估RNA质量，以RNA完整性数（RIN）≥8作为合格阈值。文库构建前通过poly-A富集对样本进行纯化，文库构建采用Illumina TruSeq Stranded mRNA试剂盒，随后在Illumina HiSeq2500平台上完成测序，测序模式为单端读长，读长50 bp，每个样本产出3000万条读段。