Overview of Pooled Library Screen
There are five primary steps required for utilizing the LentiPlex-pooled library. The following is a brief description of each step.
1. Optimization of puromycin selection conditions: To effectively eliminate colonies with no shRNA insert, optimization of puromycin selection conditions is suggested for each new cell line tested. This can be accomplished by performing an antibiotic kill curve to determine the optimal concentration of puromycin needed to eliminate untransduced cells.
The lowest concentration of puromycin that kills all untransduced cells should be utilized for subsequent experiments. Utilizing higher concentrations of puromycin may lead to unacceptably high cytotoxicity and potential off-target effects.
2. Optimization of transduction efficiency: To successfully identify shRNA sequences of interest, it is critical to set up the screen such that each cell receives only one shRNA construct. By using a low multiplicity of infection (MOI), the probability of multiple integrants per cell is greatly decreased. However, transduction efficiencies and, therefore, desired MOIs depend strongly on the target cell type.
Therefore, it is imperative that determination of the optimal MOI is carried out before starting the screen in a new cell type.
The optimal MOI can be determined by testing a range of MOIs using either Mission pLKO.1-Puro or Mission TurboGFP Control Transduction Particles on a fixed cell density.
3. Transduction and selection: After the optimal MOI is determined, cells are transduced with the viral pools. Transduced cells may be selected using puromycin before the screen is initiated depending upon the type of screen being performed. For screens utilizing a reporter enzyme, the puromycin selection step will eliminate untransduced cells, minimizing the number of cells that will need to be sorted in the end, thereby, maximizing the dynamic range of the assay. For screens assessing changes in viability, the puromycin selection step may not be required if the selective pressure is sufficient to eliminate untransduced cells.
Another variable is the length of time between transduction/
puromycin selection and application of selective pressure. Shorter times will result in fewer cells to maintain and sort at the end. Longer times will result in a broader more representative screen. In addition, the increased number of replication cycles will detect more genes that influence pathways downstream of their immediate targets. This balance will be screen-specific and depend on the type of targets that are desired at the end of the screen. It’s always important to include the most appropriate positive and negative controls for your screen.
4. PCR amplification: Total genomic DNA is isolated from the selected and expanded cell populations. The shRNA inserts can then be readily amplified using the primers provided. The provided Mission shRNA Human Positive Control Vector should be used as template in a separate reaction in order to ensure that the PCR reactions are working optimally.
5. Identification of positive hits: Sequence analysis of the PCR amplicons recovered from cells expressing the phenotype of interest can identify hits from the positive selection screen. The sequencing primer is provided in the kit.
Mission LentiPlex pooled shRNA libraries combine the RNAi Consortium’s shRNA collection with Sigma’s lentiviral manufacturing expertise to enable genome-wide RNAi screens on your benchtop. The LentiPlex pooled screening system provides enhanced delivery and long-term gene silencing in nondividing and primary cell lines. Rapid, whole-genome RNAi screens are now accessible to any researcher with minimal reagent, time, or capital equipment investment.