Cloning and Overexpression of MnSOD in mESCs

The Open Reading Frame (ORF) of MnSOD was PCR amplified from mESC cDNA using gene specific primers and cloned in BamHI and XhoI sites of pCDNA3.1 (+) (for transient transfection) and pMIG vector (for retroviral transfection) and positive clones were confirmed by sequencing (Amnion Biosciences). To clone MnSOD promoter region, 1779 bp upstream of MnSOD start site25 (link) was PCR amplified and cloned into KpnI and XhoI regions of pGL3 basic vector (Addgene).
For transient transfection, 5 µg of plasmid was transfected into confluent mESCs using XtremeGENE HP transfection reagent (Roche Diagnostics) and 24 hrs post transfection, the transfected cells were subjected to different conditions (With LIF, No LIF) and were analysed for their gene and protein expression pattern. MnSOD overexpression was also performed by retroviral transduction using pMIG MnSOD and pMIG-GFP vector control. For knockdown of MnSOD and Cu-Zn SOD, the lentiviral vectors pLKO-MnSOD shRNA and pLKO-Cu-ZnSOD shRNA (Sigma Aldrich) along with the scrambled control were transduced in mESCs. For retroviral and lentiviral production and details on transduction see supplementary material.
RNA isolation was performed using RNeasy micro kit (Qiagen) as per manufacturer's instruction. For additional details on PCR, see supporting information Experimental Procedures. Primer sequences are described in supplementary table 1.