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Figure 1. Overview of MPRA.
Oligonucleotides containing enhancer variants coupled to distinguishing tags are first generated using microarray-based DNA synthesis. The variants and tags are separated by two common restriction sites (circle/square). The oligonucleotides are PCR amplified from universal primer sites (not shown) and directionally cloned into a plasmid backbone. An invariant promoter-ORF segment is then inserted between the variants and tags by double digestion and directional ligation. The resulting reporter plasmid pool is cotransfected into a population of cells. The relative regulatory activities of the transfected variants are inferred by sequencing and counting their corresponding tags from the cellular mRNA and the transfected plasmid pool. SeeSupplementary Figure 1 for additional details.