Table 1.

Comparison of screen stages for shRNA and siRNA screens

Screen stage	shRNA	siRNA
1. Cell-based optimization	Define MOI for cell line	Establish transfection conditions
	Identify +ve, −ve controls	Identify +ve, −ve controls
	Make virus for individual shRNAs	siGLO and nontargeting control
2. Assay development	Establish screen phenotype	Define assay parameters
	Verify phenotype of controls in dilution of pools	Robustness, Z′ factor
		Develop automation
		Identify analysis rules
3. Screen	Transduce cells with library pool	Primary SMARTpool screen
	Select with GFP/puromycin	Duplicate or triplicate technical replicates
	Assay: select reference population	Analysis ongoing
	Freeze cells, extract genomic DNA
	Amplify gDNA and NGS analysis
4. Bioinformatics	Process NGS data	Statistical analysis
	Statistically rank shRNAs	Define hit list
5. Validation	Identify individual shRNA hits	Secondary validation screen
	Make virus for all constructs per target	Deconvolute SMARTpools
	Rescreen using same assay	Same assay or different assay
	Verify knockdown	Additional cell lines
6. Bioinformatics	Pathway analysis	Pathway analysis
	Data mining	Data mining
		Tertiary analysis, more cell lines, different assays

shRNA and siRNA screen stages broken into chronological order. Stages can take different times depending on the assay and quantitation method. Bioinformatics analysis is an ongoing effort that intervenes in the screen process at several points. Central to each screening platform is identification of robust positive (+ve) and negative controls (−ve). For the siRNA platform, siGLO, a fluorescent reporter, is used to indicate transfection efficiency.