Successful pooled shRNA screening requires a high shRNA fold representation
Each step of the pooled screening workflow, from transduction to hit identification, has been empirically tested. Amplification conditions were identified to ensure uniform amplification and high reproducibility (Strezoska et al. 2012). The pooled shRNA screening workflow relies on Illumina-adapted PCR primers for identification of shRNA hairpin sequences from gDNA by high-throughput sequencing on Illumina instrumentation. Primer pairs are optimized for efficient amplification of the SMARTvector shRNA or shMIMIC microRNA while minimizing thermodynamic bias and variation in representation. In addition, the PCR primers have built-in adaptor and index sequences that allow the researcher to easily move from PCR amplification to Illumina high-throughput sequencing. Direct identification of shRNA passenger strand facilitates data analysis and ensures accurate target gene identification.
PCR amplification and Illumina high-throughput sequencing workflow.
Illumina-adapted primers and Phusion Hot Start II High-Fidelity DNA Polymerase are used to PCR amplify integrated shRNA sequences and add Illumina flow-cell binding sequences. The resulting amplicons are run on Illumina platform sequencers, using the sequencing primers provided. shRNAs that are enriched or depleted during the screen are identified as hits, and the genes that they target are identified. Hits can be confirmed and studied further using individual SMARTvector Lentiviral shRNAs and shMIMIC Lentiviral microRNAs.
Utilize optimized and experimentally validated PCR primers for NGS anaylsis
SMARTvector Indexing PCR and Sequencing Primer Kits include optimized and experimentally validated primers, enabling:
High-level multiplexing; each primer kit contains 12 unique index primers, 24 in total
Efficient PCR amplification of genomic DNA with minimal bias
High-throughput multiplexed sequencing for hit identification