design: STAT3 expression is reduced in cardiac pericytes in HFpEF and its loss reduces cellular adhesion and induces pericyte senescence
library_construction_protocol: STAT3 silencing was performed using siRNA (Sigma; sequences: GGAUAACGUCAUUAGCAG, UCUGCUAAUGACGUUAUCC. Concentration, 100µM). hPL-PC were seeded with a density of 77.000 cells/well in a 6-well-plate 24 h before transfection. STAT3 siRNA and control siRNA were used at a final concentration of 50 nM. For each well an independent solution, containing siRNA diluted in 200 µl OptiMEM medium (51985034, Gibco) was prepared. Additionally, 5 µl Lipofectamine RNAiMAX transfection reagent (13778150, ThermoFisher) was diluted in 195 µl OptiMEM. The siRNA diluted in OptiMEM was added to the Lipofectamine mix and incubated for 15 min at room temperature. Cells were washed with 1 ml OptiMEM medium per well. Then, 1.6 ml OptiMEM medium was added per well and the transfection mix (400 µl) was added dropwise. After 4 h incubation the medium containing the transfection reagents was removed, and cells were further cultured in Pericyte Growth Medium 2 (C-28041, PromoCell) as indicated above. Cells were transfected as indicated above and RNA was isolated using RNeasy Plus Mini Kit (74136, Qiagen) according to manufacturer's instructions. For whole-transcriptome analysis ribosomal RNA (rRNA) was removed from a total amount of 250 ng RNA per sample followed by cDNA sequencing library preparation utilizing the Illumina® Stranded Total RNA Prep, Ligation with Ribo-Zero Plus kit (Illumina) according to the manufacturer's instructions.