3. Procedure Overview

Quantifying the representation levels of sgRNA, shRNA, or barcode sequences in experimental samples derived from genetic screens using pooled effector libraries first requires isolation of the genomic DNA. After DNA isolation, effector or barcode sequences are amplified by PCR (First Round PCR) from purified genomic DNA using library-specific primers. A Second Round of PCR amplification of the effector and barcode sequences is then performed with “nested” primers to introduce indexes and generate template compatible with Illumina NGS. Finally, the representation of sgRNA or barcode sequence in each sample are quantified by counting the number of specific reads generated by NGS on an Illumina Instrument (e.g., NextSeq, NovaSeq, HiSeq, or MiSeq).

Note: The protocol was optimized using a Life Technologies Veriti® Thermal Cycler. Use of other PCR enzymes and/or thermal cyclers may require additional optimization.