Transfection of cells with siRNA, miR, and anti-miR

IMR90 cells, HPrEC and NSC were transfected with 20-30 nM siRNA (Qiagen), miR or anti-miR (Ambion) in 96-well or 6-well plates. A 3.5% solution of HiPerFect transfection reagent (QIAGEN) was prepared in serum-free medium and then mixed with the siRNA. The mix was incubated for 30 min at room temperature and then added to the cells. The cell culture medium was replaced the following day and cells were either fixed for immunofluorescence (IF) or harvested for RNA extraction 48-120 h later. The Cy™ 3-labelled siGLO™ cyclophilin B siRNA (Dharmacon) was used to monitor transfection efficiency. Sequences specifying shRNAs against human NR2E1 were cloned in pRetroSuper as previously described^{12 (link)}. Lentiviral pLKO-based shRNA targeting mouse NR2E1 (pLKO-shNR2E1.4 and pLKO-shNR2E1.4) were obtained from Sigma (TRCN0000026019 and TRCN0000026030). The sequences of siRNA and shRNAs used are listed in Sup Table 3. Knockdown of p16^INK4a, and CBX7 was achieved using validated shRNA constructs^{2 (link), 28 (link)}.