TITLE: Genetic Modification of Human Induced Pluripotent Stem Cells (hiPSCs) by Lentiviral Transduction Protocol
AUTHORS: Roshni Jaffery, Ningbo Zheng, Jiakai Hou, Ashley Guerrero, Si Chen, Chunyu Xu, Nicholas A. Egan, Ritu Bohat, Weiyi Peng

[DESCRIPTION]
This is the protocol for the genetic modification of human induced pluripotent stem cells (hiPSCs) using lentiviral transduction method.

[STEPS]
SECTION: Day 1
1. Grow and culture 293T cell line in 10 cm plates.
SECTION: Day 1
2. Grow and culture hiPSCs in pre-coated VTN 6 well plates.
SECTION: Day 1
3. When there are enough 293T cells, seed 5 million 293T cells a 10cm plates in 10mL DMEM
complete media per plasmid of interest.
SECTION: Day 1
4. Grow overnight at 37 deg C and 5% CO2 until 80%-90% confluence.
SECTION: Day 2:  Transfection
5. Change media of wells. Make a master mix in 1.5 mL Eppendorf tubes for each plate as follows:
 
 
 
SECTION: Day 2:  Transfection
6. Vortex 10s.
SECTION: Day 2:  Transfection
7. Incubate 10 min RT *NEVER OVER 30 MIN*.
SECTION: Day 2:  Transfection
8. Add all of mix to wells dropwise and rock plate and incubate overnight 37 deg C and 5% CO2.
SECTION: Day 3
9. Check cells 24 hours post-transfection. 
SECTION: Day 3
11. 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.
SECTION: Day 3
10. 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.).
SECTION: Day 4: Virus Collection
12. Check cells 48 hours post-transfection.  Collect virus in 50 mL tube.
SECTION: Day 4: Virus Collection
13. Centrifuge 1500 rpm for 5 minutes.
SECTION: Day 4: Virus Collection
14. 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
SECTION: Day 4: Virus Collection
15. Concentrate 1:40 [~42-45mL of diluted virus makes approximately 0.8-1mL of
concentrated virus] 
SECTION: Day 4: Virus Collection
15.1. Collect and label MilliporeSigma‱ Amicon‱ Ultra-15 Centrifugal Filter Units.
SECTION: Day 4: Virus Collection
15.2. Rinse column with PBS and spin 2,000 × g for 5 min.
SECTION: Day 4: Virus Collection
15.3. Add up to 10 mL of sample to the Amicon‱ Ultra filter device.

SECTION: Day 4: Virus Collection
15.6. Collect concentrated virus and aliquot 0.2mL per Eppendorf tube. Use virus, other tubes store -80degC for future use if necessary.
SECTION: Day 4: Virus Collection
15.5. o  
Repeat adding rest of virus to Amicon‱ Ultra filter device and centrifuge again at 2,000 × g maximum for 10 minutes until all virus is concentrated.

SECTION: Day 4: Virus Collection
15.4. Centrifuge at 2,000 × g maximum for 10 minutes.
SECTION: Day 4 (continued): Virus infection
16. Remove the culture medium from cells, add 850uL fresh Stemflex complete medium into
each well of 6 well plate.
SECTION: Day 4 (continued): Virus infection
17. Add 150ul virus and 1ug/ml polybrene into cells. Rock plate.
SECTION: Day 4 (continued): Virus infection
19. Incubate at 37 C0, 5% CO2 overnight.
SECTION: Day 4 (continued): Virus infection
18. Centrifuge at 1000rcf for 90min at room temperature.
SECTION: Day 5: Change medium
20. Replace the medium with 2ml fresh Stemflex complete medium.
SECTION: Day 6: Check cells
21. Check the expression of plasmid via microscopy (if applicable).
SECTION: Day 6: Check cells
22. Begin selection (if applicable).
SECTION: Day 7 and onward
23. Check the cell status.
SECTION: Day 7 and onward
25. Freeze cells to store.
SECTION: Day 7 and onward
24. Split cells if necessary.