Mitochondrial DNA base editing in HEK293T cells

Plating of

The day before transfection, trypsinize and count the cells.

In a , plate 150000 cells per well in 1mL per well of complete HEK media ( with 10% volume without antibiotics).

Wait for cells to attach at 37°C in a 5% CO₂ tissue culture incubator.

Transfection of DdCBE Plasmids

For each well of cells to be transfected, dilute 3µL of into 50µL (total volume) of and mix well.

In a separate tube, for each well of cells to be transfected, dilute 2µg of each DdCBE plasmid or with that had been modified to include PuroR marker (left and right, for total of 4µg plasmid DNA (pDNA))

into 50µL (total volume) of . Then add 8µL P3000 Reagent from (a 2:1 ratio to DNA) directly to the diluted pDNA. Mix well.

Add the diluted pDNA solution in P3000 reagent (from 2.2) to diluted (from 2.1), mix, and incubate for0h 15m 0s min at room temperature.

Discard the old medium in the well. Add 1mL complete HEK media ( with 10% volume without antibiotics) to each tube, mix well, and add to the corresponding well. Do this step well by well. Incubate the cells at 37°C in a 5% CO₂ tissue culture incubator.

Selection of the Transfected Cells

18h 0m 0s hrs later, replace the medium with complete HEK media ( with 10% volume without antibiotics) containing up to 5ug/mL of and 1ug/mL of

Continue selection for 10-14 days and replace the medium every 2 days with complete HEK media containing and . Ensure drug selection is not finished until all the control untransfected cells are dead. Passage the cells to a larger well-size or flask if needed.

Once selection is finished, maintain the cells in complete HEK media ( with 10% volume without antibiotics) for at least 2 days before performing any experiments.