Illumina HiSeq 1500 paired end sequencing; RNA-seq of human fibroblast cell cultures treated with resveratrol against DMSO treated controls, with and without siRNA treatment targeting SIRT1
RNA-seq of human fibroblast cell cultures treated with resveratrol against DMSO treated controls, with and without siRNA treatment targeting SIRT1
Cells were harvested 24 hours after treatment with resveratrol. Fibroblasts were cultured in 12-well plates at 37 oC, in 5% CO2, in RPMI1640 medium with 10% fetal calf serum, 1% penicillin/streptomycin, and 1% L-glutamine. Knockdown of SIRT1 was achieved by transfection with SIRT1 siRNA (LQ-003540-00; Dharmacon, Boulder, US) at 30 nM using Lipofectamine transfection reagent. Knock-down was assessed by western blot probed with anti-SIRT1 antibody (sc-15404, Santa Cruz, CA, US) and anti- β-actin antibody (ab8229, abcam, Cambridge, UK). Purified resveratrol (RSV) (R5010; Sigma-Aldrich, MO, US) was used at 75µM at 48h after siRNA transfection, it was dissolved in DMSO and diluted in RPMI1640. Basecalls were made using Illumina's Casava software Total RNA was extracted with Isol-RNA lysis agent (5PRIME GmbH, Hamburg, DE). RNA purity, integrity, and concentration were determined using an Agilent 2100 Bioanalyzer (Agilent Technologies Inc., CA, US) and only RNA with a RIN value of 8.0 or higher and a 28s/18s ratio above 1.8 was used for sample preparation. The libraries were prepared according to Illumina TruSeq RNA Sample Prep Kit protocols, quantified with KaPa Library quantification Kits (Cat KK4824; KaPa Biosystems, Roche, CH).
Experimental Factor: phenotype
SIRT1 knockdown
Experimental Factor: compound
vehicle