PCR amplification, quantitative PCR and Illumina sequencing schema. (a) Diagrammatic representation of the complete integrated shRNA construct. LTR, long terminal repeat; Ze, zeomycin resistance bacterial selectable marker; tGFP, turbo GFP; IRES, internal ribosome entry site; Puro, puromycin mammalian selectable marker; RRE, Rev response element; sinLT, self-interacting LTR. (b) The structure of the shRNAmir construct. The sense and antisense shRNA sequences hybridize to form a hairpin loop structure. (c) PCR primer alignment to the shRNA construct. The PCR primers incorporate p7 and p5 sequences to enable capture on an Illumina flowcell. (d) Sequencing primer, quantitative PCR (qPCR) primer and qPCR dual label probe alignment to the shRNA PCR product. CMV, cytomegalovirus; DLP, dual-labeled probe.