Table 1.

Example of Pooled Short Hairpin RNA Screen Protocol

Step	Parameter	Value	Description
1	Seed cells	1 mL	Cells in media/polybrene solution (4 mg/mL final concentration)
2	Transduce with lentivirus library	50 μL	Spin infection: 2,000 rpm for 1 h at 37°C
3	Pool replicates in T225 flasks	50 mL	Let cells sit at RT for 20 min before placing them in an incubator
4	Select for infection	4–5 days	Puromycin selection for integration of lentiviral vector
5	Infect with virus of interest	100% infection	Allow to proceed for appropriate length of time for chosen readout
6	Select uninfected cells	Various readout	FACS or cell viability
7	Sequence lentiviral vector	NGS	Illumina sequence the processed lentiviral vectors

Step Notes

1. Want sufficient cell representation to maintain at least 500 lentiviral-transduced cells per perturbagen in the pool. Polybrene concentration predetermined for your cell type.

2. The amount of virus to infect ∼30%–50% of the cells must be predetermined.

4. Add puromycin after cells have settled in flasks; appropriate concentration for your cell line predetermined.

5. For fluorescent sorting or cell viability assay, the length of time predetermined for your virus/cell line combination.

6. FACS: sort for fluorescent-reporter low cells; cell viability: collect live cells after allowing CPE to proceed.

7. Extract genomic DNA, PCR process lentiviral vectors, sequence the shRNA insert by NGS.

CPE, cytopathic effect; FACS, fluorescence activated cell sorting; NGS, next generation sequencing; shRNA, short hairpin RNA.