Human iPSC transfection
All plasmids were purified using the Qiagen Endo-free Plasmid Maxiprep kit. Plasmids were nucleofected into iPSC cells using the Lonza 4D-Nucleofector X unit (Buffer P3, Program CB-150) according to manufacturer\\\\\\\'s instructions. For each 20 µl nucleofection reaction, 0.2–0.5 × 106 iPSC were transfected with up to 4 µg of plasmid DNA. Post-nucleofection, iPSC were plated onto 24- and 96-well Matrigel-coated plates containing mTesr1 media plus 10 µM Y-27632.
For CRISPR-based nucleofections with a targeting vector (Figures ?(Figures11 and ?and2,2, and Supplementary Figures S2–S7), 2 µg of targeting vector plasmid, 0.5 µg of Cas9 plasmid, and 1.5 µg of total sgRNA plasmid were used. When two sgRNAs were used, 0.75 µg of each plasmid was combined. When no sgRNAs were used, 1.5 µg of pUC19 was used instead.
For CRISPR-based nucleofections without a targeting vector (Figure ?(Figure33 and Supplementary Figures S10 and S11), 2 µg of total plasmid was used: 0.5 µg of Cas9 plasmid with 0.75 µg of each sgRNA plasmid or pUC19.
For TALEN-based nucleofections with a targeting vector (Supplementary Figure S5), 2 µg of targeting vector plasmid plus 2 µg total of TALEN plasmid was used. For one dsDNA break using one TALEN pair, 1 µg of each TALEN-expressing heterodimer plasmid was used. For two dsDNA breaks using two TALEN pairs, 0.5 µg of each TALEN-expressing heterodimer plasmid was used.