Arrayed Virus Production Protocol (v1.0 03-01-2023)


Required reagents (not included in starter kit)
	HEK293FT cells (Thermo Fisher Scientific #R70007)
	OptiMEM (Thermo Fisher Scientific #31985070)
	Polyethylenimine PEI transfection reagent (Polysciences #23966)
	DMEM High Glucose (Cytvia #SH30243.01)
	Bovine Serum Albumin (BSA) (VWR #AAJ65097-18)
	Syringe Filter, 0.45μm, Sterile (CELLTREAT #229745)
	pMD2.G plasmid (Addgene #12259)
	psPAX2 plasmid (Addgene #12260)


Note, this protocol is not optimized handling for large numbers of individual viruses in an arrayed format.
If it is desired to produce large numbers of individual viruses instead of pooled virus production, 
we suggest to scale down the reactions to enable plate-based processing with multichannel pipettes.

Tip:
	a. We use PEI as a transfection reagent instead of Lipofectamine.


6-well format transfection

	- Plate 1*10^6 HEK293FT cells per 6-well in 2mL DMEM+10%FBS growth medium.
	  Depending on cell counter the cell number may be adjusted.
	  Ideally, cells are 95-100% confluent at the time of transfection.
	- Let cell adhere over night (up to 24 hrs) till they reach the desired confluency.
	- Set up 2 master mixes
		MasterMix1
			optiMEM 	50 µl
			PEI 		7.5 µl (note, this may need to be optimized depending on your PEI stock. We use a 3:1 ratio of PEI to plasmid micrograms) 

		MasterMix2
			optiMEM 	50 µl
			pMD2.G 550ng	x µl
			psPAX2 825ng	y µl
	- Mix MasterMixes by vortexing. 
	- Distribute Mastermix2 (50 + x + y µl) per reaction into sample tubes.
	- Add a X µl (1125ng) of the pLentiRNAGuide_003 plasmid of interest to each sample tube
	- Mix (vortexing)
	- Add 57.5µl MasterMix1 mix to each sample tube.
	- Mix each sample by pipetting up and down 15-20 times.
	- Incubate for 10 minutes at room temperature.
	- In the meantime, take out the 293FT cells and remove part of the medium from each well before transfection (here leave cells with 1ml).
	- Add the PEI-mix (~110µl) dropwise to the cells distributing it across the entire surface area.
	- Once PEI-mix has been added to each well in the plate, mix by swirling the 6-well plate in an 8/infinity-shape ~10 times.
	- 6-8 hours post transfection (or the next morning) carefully remove all the media in the well and replace with 2ml DMEM+10%FBS+1%BSA (sterile-filtered).
	- After additional 48hours, harvest the virus supernatant.
	- Transfer the 2ml into a 2ml tube and spin for 4 min at 1000g at 4°C.
	- (Optional: sterile filter using 0.45µM filters. Recommended for single-cell experiments.)
	- Transfer the top 2x800ul into sterile Eppendorf tubes and freeze @ -80°C for storage till usage. Freeze two aliquots to minimize free-thawing cycles.
	  (Freezing and thawing may slightly reduce viral titer, but ensures that no viable cells will be carried over)

Note, viral titer of pLentiRNAGuide_003 is typically high.
Usually using 50-100µl of viral supernatant will lead to a multiplicity of infection of about 0.2-0.3 MOI, in susceptible cells (e.g. HEK293 or K562 cells).
Further, pLentiRNAGuide_003 plasmids contain a GFP-2A-Puromycin cassette. 
Therefore, viral titer may be determined by qunatifying %-survival upon puromycin challenge, or by flowcytometry measuring the fraction of GFP+ cells in unselected cell populations.
Typically, infected cells may be selected with Puromycin to select for guide RNA expressing cells.