P3 primary cells nucleofection kit

Plasmids pSF-tdTomato-END for NHEJ repair and pSF-tdTomato-HOM for SSA repair (Fig 1A) were digested with XhoI and ApaI and complete double-digestion was verified for quality control as described previously [34 (link)]. For all cell types investigated, DSB repair assays were performed using 400ng of either END (for NHEJ) or HOM (for SSA) linearized plasmids transfected into cells using the P3 Primary cells nucleofection kit on the 4D Nucleofector system (Lonza). Electroporations were performed using the 20μl format (strips of electrocuvettes) and the EO-115 program. Cells were then recovered in 180μl of RMPI 1640 with 10% FBS in a 96-well plate and placed in an incubator for 12h. To determine repair efficiency, cells were then resuspended in HBSS containing 0.3μg/ml DAPI and the proportion of EYFP⁺ cells among viable (DAPI negative) transfected cells (tdTomato⁺) determined using a BD LSRFortessa cell analyzer with the FACSDiva software (version 6, BD Biosciences) as described previously [34 (link)]. NHEJ and SSA repair were analyzed in parallel and in triplicates for each sample. The number of technical replicates (separate transfections of the same cells with the same construct in the same experiment) was in some cases reduced to 1 or 2 for patient samples, based on the number of lymphocytes available.

Both GM01953 cells and primary lymphocytes were transfected with the P3 primary cells nucleofection kit (Lonza) using the 96-E0-115 program recommended for “high functionality” transfection of uninduced T-cells on the Amaxa Nucleofector 96-well Shuttle system. Typical transfections used 150,000–190,000 GM01953 or 125,000–200,000 primary lymphocytes (plus a variable amount of other WBCs depending on the sample preparation method). One individual aliquot of frozen GM01953 or primary lymphocytes was used for each experimental day to generate 3 measurements of each type of repair (NHEJ and SSA) plus all controls for the FACS analysis (1–3 measurements depending on the experimental day for GM01953).