Protein knockdown by siRNA interference

For depletion of CtIP, RAD52 and RAD51, a pool of three siRNAs, specific for each protein, as previously described (Kostyrko et al., 2017) (Eurogentec), was introduced by electroporation (Nucleofector^™ II device, Amaxa Biosystems) following manufacturer’s protocol and program U-23. Briefly, the following oligonucleotides were used for depletion of CtIP: 5′-GUG​CAA​GGU​UUA​CAA​AUA​A-3′; 5′-CAA​AGU​CCC​UGC​CAA​ACA​A-3′; 5′-AGA​AUA​CUC​UCC​AGG​AAG​A-3′ (Eurogentec). Similarly, for downregulation of RAD52, again a cocktail of three specific RNAs was utilized, following the same transfection procedure: 5′-UGA​GAU​GUU​UGG​UUA​CAA​U-3′; 5′-ACU​GCA​UUC​UGG​ACA​AAG​A-3′; 5′-CCC​UGA​AGA​CAA​CCU​UGA​A-3′. For efficient RAD51 ablation the following three specific siRNA sequences were used: 5′-GUG​CCA​AUG​AUG​UGA​AGA​A-3′; 5′-GGG​AAU​UAG​UGA​AGC​CAA​A-3′; 5′-GGC​GUU​CAG​AAA​UCA​UAC​A-3′. The following negative control RNA (ncRNA) sequence was used: 5′-UUCUCCGAACGUGUCACGUdTdT-3′. ncRNA is used for mock-transfection.