Bulk RNA seq of WT and TSC2KO mouse mesenchymal vascular progenitors

To elucidate potential cell-based mechanisms by which deregulation of mTOR signaling in MVPC drives microvessel rarefaction and afore mentioned loss of tissue structure, we isolated eGFP positive MVPC lines by flow sorting and analyzed their transcriptomic signatures. Unbiased transcriptomic comparison was used to define mechanisms underlying the decreased stemness and angiogenic ability in Tsc2KD mTOR activated versus WT MVPC. Additionally, we identified a Tsc2KD line that exhibited pS6 expression similar to WT levels suggesting cell intrinsic adaptive regulation of mTOR. Bulk RNA seq was performed in triplicate using primary WT, Tsc2KD mTOR regulated and Tsc2KD mTOR activated (mTOR+) lines. Following normalization, hierarchical clustering analyses were presented as a heatmap depicting significant differences in gene expression between WT and Tsc2KD lines. Tsc2 KD was confirmed in bulk RNA sequencing analysis of the MVPC lines Overall design: mRNA (Qiagen kit/column) Poly A selected total RNA Lib/Bulk RNA seq; NOVA seq 6000 Paired End 150 cycle 2x150; 80 million PE reads; Nugen library kit/prep; 100ng mRNA (polyA selected) in 50uL or less (water or TE)

为阐明微血管周细胞（MVPC）中雷帕霉素靶蛋白（mTOR）信号通路失调驱动微血管稀疏及前述组织结构丧失的潜在细胞机制，我们通过流式分选分离出增强型绿色荧光蛋白（eGFP）阳性的MVPC细胞系，并分析其转录组特征。我们采用无偏倚转录组比较分析，明确了Tsc2基因敲低（Tsc2KD）且mTOR激活组相较于野生型（WT）MVPC的干细胞特性降低及血管生成能力下降背后的分子机制。此外，我们筛选到一株Tsc2KD细胞系，其磷酸化S6蛋白（pS6）表达水平与WT组相近，提示存在细胞内在的mTOR适应性调控机制。我们对野生型原代细胞、Tsc2KD且mTOR受调控组、Tsc2KD且mTOR激活组（mTOR+）的细胞系进行了三次重复的批量RNA测序（bulk RNA-seq）。标准化处理后，我们通过层级聚类分析生成热图，直观展示WT与Tsc2KD细胞系间显著的基因表达差异。MVPC细胞系的批量RNA测序分析验证了Tsc2敲低效率。实验整体设计如下：采用Qiagen试剂盒/吸附柱提取mRNA，经polyA富集的总RNA构建文库后进行批量RNA测序；使用NOVAseq 6000平台进行双端150循环测序（2×150）；每个样本获取8000万条双端reads；采用Nugen文库制备试剂盒进行文库构建；起始投入量为100ng经polyA富集的mRNA，总体积不超过50μL（溶剂为水或TE缓冲液）。