Figure 2. Representative histogram for pooled shRNA library representation showing that >95% of constructs are within 5-fold of the mean.

Importance of equimolar distribution in a pooled shRNA screening library

Changes in the relative distribution of the shRNA in a pool indicate a selective advantage or disadvantage during the screening process and are detected using next-generation sequencing (NGS).  To ensure that artificial changes in population are not introduced, pools should be constructed to minimize variation in representation across hairpins. For example, false negative results can occur when an shRNA is under represented in the population and lost by chance due to low representation during transduction, insufficient cell plating during passaging, or insufficient sample size. 

transOMIC pooled shRNA libraries start with sequence-verified arrayed shRNA resulting in better normalization of shRNA representation in pools. Typical pooled libraries have a relatively narrow range of shRNA distribution across the pool. >95%  of the shRNA are within a 5-fold of the mean (Figure 2).

Choice of promoter for hard to transduce cells

Mammalian promoters may differ in expression level or be silenced over time depending on the target cell line.  Variation in expression level can affect fluorescent marker expression as well as knockdown efficiency.  The Spleen Focus Forming Virus (SFFV) LTR provides a functional promoter in a number of human and mouse cell lines and is standard in the ZIP lentiviral vector used to create pooled shRNA screening libraries ^3,4,5.  However, for cell lines where a different promoter may be optimal, the ZIP promoter testing kit (TLN0005) is available with a panel of vectors to quickly test for optimal expression in target cell lines.  The ZIP lentiviral vector is available with three different promoter options (SFFV, human CMV or murine CMV) driving expression of shRNA (see schematic on the right). The fluorescent marker and shRNA are on the same transcript allowing the level of fluorescence in the cell to be used as a direct indication of shRNA expression through visual inspection. Ready-to-use viral particles for each of the three vectors are provided for transduction in target cell lines of interest to determine the optimal promoter for efficient shRNA expression.

The ZIP promoter selection kit (TLN0005) provides sufficient viral particles of a non-targeting shRNA in each vector to transduce the target cell line and determine the optimal promoter.