Pcbascei

For determining the HR and NHEJ proficiency in WT and Rif1^−/− cells the following plasmids were used: pDRGFP (Addgene plasmid #26475) and pCBASceI(Addgene plasmid #26477) for HR and pCVL Traffic Light Reporter1.1(Sce target) (Addgene plasmid #31482) along with pCBASceI for NHEJ. For measuring and controlling transfection efficiency, Turbo-GFP expressing plasmid (Sigma, MISSION SHC003) and Scrambled plasmid (Sigma, MISSION SHC002), were used. In all, 2.5 x 10⁵ cells were co-transfected with 3 μg of plasmid combinations (i.e., 1.5 μg of each plasmid) using Xtremegene-9 reagent from Roche in six-well dish. Cells were transfected twice at an interval of 24 h. Post 48 h of transfection, cells were harvested and the GFP-positive cells (for HR) and RFP-positive cells (for NHEJ) were assessed by flow cytometry. Fifty thousand events were recorded for each sample. Background normalization was done by using samples co-transfected with scrambled—reporter plasmid and also scrambled—pCBASceI plasmid. Final percentage of GFP and RFP-positive cells were calculated based on the transfection efficiency of the cells. Each experiment was independently performed at least thrice.

T24 cell received the stable transfection by employing with pimEJ5GFP (AddGene 44026). In brief, pimEJ5GFP received the digestion by exploiting XhoI, as well as the transfection to T24 cell. Steady transfected cells received the selection and maintenance within media covering 2 μg/ml puromycin. To observe DSB localization, stable shNC and shTEX10 cell with the EJ5 reporter received the transfection by using pCBASceI (AddGene 26477), the 12 h incubating process to allow for plasmid expressions and subsequently the observation of DSB localization. To measure NHEJ efficiency, stable shNC and shTEX10 cell with the EJ5 reporter received the transfection by using pCDH and XRCC6 expression plasmids, then the 24 h incubating process for enabling plasmid expressions. Cell received the transfection by using pCBASceI (AddGene 26477), expressing I-SceI, and the 24 h incubation. Next, cell received the harvesting process, the washing process by using PBS, and the resuspending process within PBS. GFP received the detection based on cell sorting under the activation from fluorescence (FACS Calibur, Becton-Dickinson).

pDRGFP (plasmid # 26475, Addgene, Watertown, MA, USA) and pCBASceI (plasmid # 26477, Addgene) plasmids used for the HRR assay were a kind gift from Maria Jasin [46 (link),47 (link)]. Equimolar quantities of both plasmids were used for co-transfection of the cells using Lipofectamine 3000 reagent according to the manufacturer’s instructions. The cells were collected by trypsinization, washed in PBS then re-suspended in 500 µL of PBS. A total of 50,000 cells were analysed by flow cytometry to estimate the number of GFP-positive cells. The % HRR activity was calculated by subtracting the % of GFP-positive cells in untransfected from the co-transfected sets.