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 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)