TITLE: LENTIVIRAL TRANSDUCTION OF HUMAN PLURIPOTENT STEM CELLS
AUTHORS: Renuka Ravi Gupta, Nona Farbehi, hendersa, Vikram Khurana, Gist Croft, Lorenz Studer, Joseph Powell

[DESCRIPTION]
We have developed a protocol for lentiviral transduction of human pluripotent stem cells (hPSCs), including induced pluripotent stem cells (iPSCs) or human embryonic stem cells (hESCs). In this protocol, concentrated lentiviral supernatant with a Multiplicity Of Infection (MOI) ranging from 0.1 to 0.3 (equivalent to 10% to 30% Blue Fluorescent Protein (BFP) positive cells) is combined with E8 Flex media and added to adherent H9 CRISPRi dCAS9 cells in 48-well plates. Subsequent centrifugation (spinfection) is employed to ensure efficient transduction. Transduction efficiency is assessed by determining the percentage of cells expressing Blue Fluorescent Protein (BFP) using Fluorescence Activated Cell Sorting (FACS).

[STEPS]
SECTION: Day 0: Coating wells with VTN-N and seeding hPSCs
1. Add 60 ul of VTN-N to 6 ml of DPBS.
SECTION: Day 0: Coating wells with VTN-N and seeding hPSCs
2. Coat 100 ul per well in a 48 - well plate.
SECTION: Day 0: Coating wells with VTN-N and seeding hPSCs
3. Incubate the plate at room temperature for an hour and the plate is ready to be used.
SECTION: Day 0: Coating wells with VTN-N and seeding hPSCs
4. Seed 3x104 cells/cm2 in a 48 well plate with E8 flex media and RevitaCell after dissociating the cells with accutase.
SECTION: Day 0: Coating wells with VTN-N and seeding hPSCs
5. Incubate the cells overnight at 37ºC with 5% CO2 and 20.9% O2.
SECTION: Day 1: Transduction of hPSCs with Lentiviral CRISPRi library supernatant
6. The H9 dCAS9 CRISPRi cells were transduced with the pooled CRISPRi library to harvest genomic DNA (gDNA), after calculating the exact volume of viral supernatant needed to achieve the desired MOI (multiplicity of infection).
SECTION: Day 1: Transduction of hPSCs with Lentiviral CRISPRi library supernatant
7. Prepare 15 ml tubes with E8 flex media and concentrated lentiviral supernatants for a MOI of 0.1-0.3.
SECTION: Day 1: Transduction of hPSCs with Lentiviral CRISPRi library supernatant
8. Add 200 ul/well of the virus media cocktail with the cells from step i.
SECTION: Day 1: Transduction of hPSCs with Lentiviral CRISPRi library supernatant
9. To increase the transduction efficiency, centrifuge the plate at 300g for 20 minutes at 25ºC.
SECTION: Day 1: Transduction of hPSCs with Lentiviral CRISPRi library supernatant
10. Incubate the cells at 37ºC with 5% CO2 and 20.9% O2 for 16-18 hours.
SECTION: Day 2: Replace media
11. Aspirate the viral supernatant media gently and immediately add maturation media.
SECTION: Day 2: Replace media
12. Return the plate back to the incubator.
SECTION: Day 3: FACs Sort
13. Aspirate the spent media.
SECTION: Day 3: FACs Sort
14. Wash the cells 10 times with DPBS to remove the viral particles from the lentivirus transduced hPSCs.
 

SECTION: Day 3: FACs Sort
15. Add 100 ul accutase and incubate the cells for 10 mins in the incubator.
SECTION: Day 3: FACs Sort
16. Note: Ideally the hPSCs should dissociate as single cells. 
SECTION: Day 3: FACs Sort
17. Neutralize the accutase with E8 flex media and collect the cells into 1.5ml eppendorf tubes. 
 

SECTION: Day 3: FACs Sort
18. Centrifuge the cells at 300 g for 4 minutes.
SECTION: Day 3: FACs Sort
19. Aspirate the spent media gently without disturbing the pellet. 
SECTION: Day 3: FACs Sort
20. Resuspend the cells in 300 ul of FACs buffer.
SECTION: Day 3: FACs Sort
21. Transfer the cells with the FACs buffer into FACs tubes.
SECTION: Day 3: FACs Sort
22. Sort the H9 CRISPRi cells to obtain 10- 30% BFP positive cells and collect the cells in E8 flex media.
SECTION: Day 3: FACs Sort
23. Centrifuge the cells at 300 g for 4 mins.
SECTION: Day 3: FACs Sort
24. Aspirate the spent media to obtain a cell pellet to be frozen or freshly used for DNA extraction.