RNA-sequencing of non-senescent and senescent mouse skin fibroblast

Mouse skin fibroblasts (MSFs) were obtained from a FASST (Fibroblasts Accelerate Stromal-Supported Tumorigenesis) mouse. This mouse model allows for spatial and temporal control for senescence induction by using a stromal specific Cre-recombinase driven by the pro-collagen-alpha II promoter. The stromal specific Cre activates expression of the p27IRESGFP transgene that is expressed from the ROSA locus. We cultured the MSFs in vitro, induced senescence using 10uM tamoxifen added to the media. Non-senescent cells were treated with equal volume of vehicle alone (ethanol). Upon tamoxifen treatment, cells were moved to a modular incubation chamber and maintained at 3% oxygen at 37 degrees celcius for 12 days total before collection. At the time of collection, cells were trypsynized and pelleted by centrifugation. The cells were lysed using Trysol reagent and RNA was isolated using a RiboPure RNA isolation kit (Ambion). Overall design: For this study, 2 treatment groups were analyzed (non-senescent, EtOH samples and senescent, TAM samples). Each treatment group was performed 3 times for a total of 6 samples for analysis. The gene expression analysis is a comparison of expression in senescent (TAM) vs non-senescent (EtOH) mouse skin fibroblasts.

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Ruhland MK, Loza AJ, Capietto AH, Luo X, Knolhoff BL, Flanagan KC, Belt BA, Alspach E, Leahy K, Luo J, Schaffer A, Edwards JR, Longmore G, Faccio R, DeNardo DG, Stewart SA