Disposable pasteur pipette

70–80% confluent iPSC were dissociated into single cells using Accutase (Thermo Scientific, cat # A1110501) and counted using a TC20 Automated Cell Counter (Bio-Rad). iPS cells (8x10⁵ per sample) were pelleted at 1000 rpm for 3 min and cell pellet were gently resuspended in 100 μl of P3 Primary Cell Solution (Lonza, PBP3–02250). Immediately prior to nucleofection, 2 μl (100 pmol/μl) of ssDO was added to 5 μl of pre-assembled Cas9/RNP. One hundred μl of iPS cells resuspended in P3 Primary Cell Solution were transferred to the tube containing Cas9/RNP complexes and ssDO. Cells were mixed twice with Cas9/RNP/Donor oligo and the mixture was transferred to the 100 μl nucleocuvette (Lonza; 2022–01). iPSC were nucleofected immediately using the ‘Primary Cell P3′ program and ‘CA-137′ pulse code. After nucleofection, iPSC were transferred using a Lonza disposable Pasteur pipette into one well of a Matrigel-coated 6-well plate containing 3 mL E8 media with 10 μM Rock inhibitors. Cells were cultured in a 32°C/5% CO₂ incubator for two days and then transferred to a 37°C incubator. Edited iPSC pools were expanded, frozen, and used for generation of clones from single cells.

iPSCs were dissociated into single cells using TrypLE (Fisher Scientific, cat #A12605036) and counted. 8×10⁵ cells were then pelleted at 1000 rpm for 3 minutes and subsequently gently resuspended in 100μl of P3 Primary Cell Solution from P3 Primary Cell 4D-Nucleofector X Kit L (Lonza, #V4XP-3024). Immediately prior to nucleofection, 2 μl (100 pmol/μl) of ssDO (Table 3) was added to 5μl of pre-assembled Cas9/RNP complex. 100μl of iPS cells, resuspended in P3 primary cell solution, was then combined with the Cas9/RNP complex and ssDO; thoroughly mixed and transferred to the 100 μl nucleofector cuvette (Lonza; #V4XP-3024). Immediately upon transfer, cells were transfected using the ‘Primary Cell P3’ program and ‘CA-137’ pulse code on Lonza Nucleofection machine. iPSCs were then carefully transferred using a Lonza disposable Pasteur pipette into one well of a Matrigel-coated 6-well plate containing 3 mL StemFlex media with 10 μM Rock inhibitors. Cells were cultured in a 32C/5% CO₂ incubator for two days and then transferred to a 37 C incubator. Edited iPSC pools were expanded and cryopreserved.