RNA-seq of human fibroblasts during pseudo-longitudinal in-vitro aging and long-term Metformin or Rapamycin treatment
RNA-seq of human fibroblasts during pseudo-longitudinal in-vitro aging and long-term Metformin or Rapamycin treatment
Cells at the Month 0 timepoint were detached upon reaching confluency using TrypLE, then pelleted and snap-frozen on dry ice, and stored at -80°C. For Months 1-6, cells were harvested after every 30 days. Primary cells were isolated from skin punch biopsies, which were immediately digested using 1mg/mL Collagenase and 1mg/mL Dispase at 37 °C overnight, before enabling outgrowth of fibroblasts on Gelatin-coated wells. Pure fibroblast cultures were generated from four donors (female, 53, 55, 67 and 68 years old) and maintained in fibroblast media containing DMEM (ThermoFisher) + 15 % fetal bovine serum (ThermoFisher) + 1x non-essential amino acids (Sigma Aldrich) + 1x antibiotic-antimycotic (ThermoFisher). Cells were expanded by detaching using TrypLE (ThermoFisher) and seeded in a 1:2 ratio.  Metformin (1mM) and Rapamycin (25nM) treatments were applied the day after harvesting Month 0 in fibroblast media. Media including compounds were freshly prepared for each media change and continuously applied to the cells throughout the 6-month culture period. Frozen cell pellets of Months 0-6 were thawed on ice, and DNA and RNA were extracted simultaneously from the same pellet using the Qiagen AllPrep DNA/RNA Mini Kit. DNA and RNA concentrations were assessed using Qubit fluorometric quantification. Quality of RNA was further assessed using the TAPE Station System (Agilent), and DNA was run on an agarose gel.  Strand specific mRNA libraries were generated using the Illumina Truseq stranded mRNA kit.