MPRA experiments were performed as previously described (Melnikov et al., 2012). Approximately 240,000 145-bp oligonucleotides representing ~11,000 distinct “tiles” with the major or minor alleles of 1,837 candidate functional variants (including 525 candidate functional variants in the CPNE1, ANGPTL3, and FRK loci) in the center, shifted by 40 nt towards the 5′ end (right-shifted), or shifted by 40 nt towards the 3′ end (left-shifted) and coupled to distinguishing barcodes were generated by microarray-based DNA synthesis (Agilent). To minimize barcode- and amplification-associated biases, each tile was coupled to ~22 distinct barcodes. Two common restriction sites separated the tiles and barcodes. The oligonucleotides were PCR amplified using universal primer sites and directionally cloned into a pMPRA1 (Addgene plasmid #49349) backbone using Gibson assembly. A minimal promoter-firefly luciferase segment from pMPRAdonor2 (Addgene plasmid #49353) was inserted between the tiles and barcodes via double digestion and directional ligation. The resulting reporter plasmid pools were co-transfected into NIH 3T3 fibroblasts using FuGENE 6 (Promega). Two independent, biological replicate MPRA experiments were performed. The relative enhancer activities of the different tiles were calculated by comparing the corresponding barcodes from the cellular mRNA and the transfected plasmid pool.
