Pooled 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)


Tips:
	a. We use PEI as a transfection reagent instead of Lipofectamine.
	b. An even representation of barcoded guide RNA plasmids in the plasmid pool should be verified before pooled virus production.
	c. If some guide RNA target genes are essential and likely to drop out from the infected cell pool over time, you can increase the relative amount of the respective plasmids in the pool to mitigate dropout. In addition, we recommend performing CaRPool-seq at an early time point (5-7days) post transduction and Doxicycline Cas13d induction. 


10cm dish format transfection

	- Plate 1*10^7 HEK293FT cells per 10cm in 10mL 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 (if >1 plate will be transfected)
		MasterMix1
			optiMEM 	300 µl
			PEI 		 60 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 	300 µl
			pMD2.G 4400 ng	  x µl
			psPAX2 6400 ng	  y µl
	- Mix MasterMixes by vortexing. 
	- Distribute Mastermix2 (300 + x + y µl) per reaction into sample tubes.
	- Add a X µl (9200ng) of the pLentiRNAGuide_003 plasmid pool to each sample tube
	- Mix (vortexing)
	- Add 360 µl 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 5ml).
	- Add the PEI-mix (~700µl) 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 10cm dish 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 10ml DMEM+10%FBS+1%BSA (sterile-filtered).
	- After additional 48hours, harvest the virus supernatant.
	- Transfer the 10 ml into a 15 ml tube and spin for 5 min at 1000g at 4°C.
	- Sterile filter using 0.45µM filters. (Recommended for single-cell experiments.)
	- Aliquot at 1 ml into sterile Eppendorf tubes and freeze @ -80°C for storage till usage. Freeze multiple 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.