Grow and culture 293T cell line in 10 cm plates.

Grow and culture hiPSCs in pre-coated VTN 6 well plates.

When there are enough 293T cells, seed 5 million 293T cells a 10cm plates in 10mL DMEM

complete media per plasmid of interest.

Grow overnight at 37 deg C and 5% CO2 until 80%-90% confluence.

Change media of wells. Make a master mix in 1.5 mL Eppendorf tubes for each plate as follows:

A	B	C	D	E
JetPRIME Buffer (Polyplus Transfection, Ref #: 114-15)	Plasmid interest	Packaging plasmid VSVG	Packaging plasmid psPAX2	jetPRIME transfection reagent (Polyplus Transfection, Ref #: 114-15)
500 uL	500 uL	2 ug	3 ug	1:2 ratio of total DNA (ug): transfection reagent (uL) =20 uL

Vortex 10s.

Incubate 10 min RT NEVER OVER 30 MIN.

Add all of mix to wells dropwise and rock plate and incubate overnight 37 deg C and 5% CO2.

Check cells 24 hours post-transfection.

First thing in the morning, exchange the media – add 15 mL pre-warmed DMEM complete media to transfected wells. (This removes the jetPRIME transfection reagent and residue plasmid.).

Split hiPSCs into clumps in StemFlex complete medium. Seed 20% confluency or ~0.1M

per well in 2 mL of 6 well plate. Note: Cell number depends on cell type.

Check cells 48 hours post-transfection. Collect virus in 50 mL tube.

Centrifuge 1500 rpm for 5 minutes.

Using a 0.45 um PDVF membrane filter and 5 mL syringe, collect virus into new 50 mL tubes. Avoid bottom of tube with cell debris

Concentrate 1:40 [~42-45mL of diluted virus makes approximately 0.8-1mL of

concentrated virus]

Collect and label MilliporeSigma^TM Amicon^TM Ultra-15 Centrifugal Filter Units.

Rinse column with PBS and spin 2,000 × g for 5 min.

Add up to 10 mL of sample to the Amicon^® Ultra filter device.

Centrifuge at 2,000 × g maximum for 10 minutes.

o

Repeat adding rest of virus to Amico^®‱ Ultra filter device and centrifuge again at 2,000 × g maximum for 10 minutes until all virus is concentrated.

Collect concentrated virus and aliquot 0.2mL per Eppendorf tube. Use virus, other tubes store -80degC for future use if necessary.

Remove the culture medium from cells, add 850uL fresh Stemflex complete medium into

each well of 6 well plate.

Add 150ul virus and 1ug/ml polybrene into cells. Rock plate.

Centrifuge at 1000rcf for 90min at room temperature.

Incubate at 37 C⁰, 5% CO2 overnight.

Replace the medium with 2ml fresh Stemflex complete medium.

Check the expression of plasmid via microscopy (if applicable).

Begin selection (if applicable).

Check the cell status.

Split cells if necessary.

Freeze cells to store.