Split iPS cells into single cells in complete medium with RVC. (500µl per well)

Seed cells at 5.0X104 cells in glass-bottom 24-well plates

Virus infection:

Remove the culture medium from cells, add ( 300µL) fresh medium without RVC into each well

Add 45µl virus and 1µg/ml polybrene into cells and mix it. Incubate in 37 degree, 5% CO2.

A	B	C	D
PLVX mCherry HLA-A2	PLVX IRES-Puro HLA-A2	pLoc Mlana

Add Polybrene into the infected well to make the final concentration to be 1ug/ml.

Stock Polybrene (10mg/mL)

Make a 100x dilution to make a working concentration of 100µg/mL

(10mg/mL) (V_stock) = (100µg/mL)(0.5mL)

V_stock= 5µL of stock into 495µL of DPBS (-)(-)

(100µg/mL) (V_stock) = (1µg/mL)(0.3mL) --> V_stock= 3µL of working concentration per 300µL of medium added

Monitor the cells for the next 48 hours

Check IRES-GFP or HLA-A2’s expression at least 48 hours after the transduction.

For antibiotic-based selection, the cells will be maintained to reform colonies and subsequently grown in medium with 1µg/mL of Puromycin to initiate selection