GSM7677243: Double knockdown treated with 915, rep 1; Rattus norvegicus; RNA-Seq
For siRNA knockdown, NRVMs were transfected with siRNA against rat Esrra (Dharmacon, M-085000-02-0005), rat Esrrb (Dharmacon, M-100126-01-0005), rat Esrrg (Dharmacon, M-100212-01-0005), rat E2f1 (Dharmacon, M-102754-00-0005) or scramble control siRNA (ThermoFisher, 4390843), using the Viromer BLUE Transfection Reagent (Lipocalyx, VB-01LB-01). Typically, 25 nM siRNA was used for NRVMs seeded in 24-well. For treatment with 915 or vehicle, cells were treated with 10uM 915 or equivalent volume of vehicle (DMSO) for 24h. Total RNA was extracted using Qiagen miRNeasy kit according to the manufacturer's instructions. Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers, followed by the second strand cDNA synthesis using dTTP, followed by end repair, A-tailing, adapter ligation, size selection, amplification, and purification. unstranded, dual indexed for paired-end sequencing 2x150