Furthermore, the requirement for maintaining the shRNA fold Butenafine hydrochloride representation throughout the experiment including the PCR amplification steps has not been addressed. In particular, the amount of gDNA input in the PCR step has not been established. Here we examined the effects of shRNA fold representation at transduction on the reproducibility of pooled shRNA screening data. We performed viability screens with a library of,10 000 shRNAs at two different fold representations and report the reproducibility of changes in proviral shRNA abundance between screening replicates determined by microarray and NGS analyses. We show that the technical reproducibility between PCR replicates from a screen can be drastically improved by 1) ensuring that the PCR amplification steps are maintained in the exponential phase and 2) using an amount of gDNA input in the reaction that maintains the average template copies per shRNA that was used during library transduction. Using these optimized PCR conditions, we also show that reproducibility between screening replicates improves with increased shRNA fold representation at transduction and amplification. This higher reproducibility results in a greater overlap of primary hits between the biological screening replicates when using either analysis method. shRNA hits with smaller fold changes in abundance were identified in screens using higher shRNA fold representation, however shRNA with robust fold changes were generally identified in screens with both low and high shRNA fold representation. The reference sample was generated by amplifying molecular barcodes from the plasmids used to create the Decode library. The test sample was generated by amplifying molecular barcodes from HeLa cells infected with the Decode lentiviral particles. HeLa cells were transduced with the Decode library at 100-fold average shRNA representation and were selected with puromycin before gDNA was isolated. Comparison of the reference and test samples from selected cells simulates a viability screen scenario where the abundance of some shRNAs has decreased in the test sample due to effects on cell viability. The correlation between replicates of each of the samples as well as their log ratios can be examined. The libraries used in pooled screening exhibit various ranges in shRNA abundance. For the reference, the shRNA abundance is expected to be a reflection of plasmid library composition. For the test sample, a larger range of abundance is expected since it depends on both library composition and biological effects from shRNAs in transduced cells. Ideally, the protocol should not introduce bias; Orbifloxacin otherwise the changes detected in shRNA abundance might not be biologically relevant. Therefore, we examined the effects of PCR amplification on the range of shRNA abundance for reference plasmid and the transduced test sample by looking at the minimum fold difference between the least and most represented shRNAs for 70% of the shRNA population. Similar to the Pearson correlation values, we find that the abundance range increases for both samples with increased PCR cycle number and decreased template copies per shRNA. The fold change in shRNA abundance is particularly pronounced for the transduced test sample, where the difference 
between the least and most represented shRNAs changes from 14- to 69-fold. For this reason the impact of PCR amplification on pooled shRNA screening data reproducibility needed to be systematically examined further. First, the number of amplification cycles during PCR was examined because amplification is most quantitative during the exponential phase of the reaction where the copy number is doubled at each cycle for 100% efficient reactions. Second, the amount of gDNA input used for PCR was examined.