ProliHHs electroporation

Electroporation was performed when ProliHHs reached the 80%-90% of confluence in a dish. Two days before the electroporation, cells were added AAV2.7m8-donor virus at an MOI (GCs/cell) of 100,000 and then incubated at 37 °C overnight. For preparation of targeted integration in ProliHHs, the procedure was conducted following the manufacturer’s instructions using a Lonza 4D electroporation system. 3ml EHM media (Remove the Penicillin-Streptomycin from the EHM medium to prevent cell death caused by the presence of Penicillin-Streptomycin after electroporation) per well of a 6-well plate was prewarmed. After two days of AAV infection, the cells were digested and counted. The digested cells were washed with PBS and centrifuged at 3×105 ProliHHs in an EP tube. The PBS was then completely removed. After preparing the RNPs following the aforementioned method, the transfection solution for suspension cells was prepared using the Lonza P3 Primary Cell 4D-Nucleofector X Kit S (32RCT, Cat. No. V4XP-3032). 18µl Nucleofector Solution and 4µl Supplement Solution were mixed thoroughly before use. The mixed transfection solution was then added to the cells and the RNPs were added to the cell suspension using a 20µl pipette tip. The cell suspension was gently pipetted to ensure thorough mixing. The cell-mixture was transferred into an electroporation cuvette plate. Electroporation program DS198 was chosen for electroporation. After electroporation, cells were moved out of the electroporation cuvettes and gently supplemented with 37°C prewarmed medium in 6-well plate.