Enhancer function reporter assay

The following vectors were used in this protocol: pGL3 luciferase reporter (Promega), pGL4.74[hRluc/TK] control vector (Promega). ~200–300 base pair non-coding regions of RGS18, ADRA2A and PEAR1 and the associated alleles surrounding the various SNP variants were cloned into the pGL3 luciferase vector. We created two modified constructs to assess functionality of the various loci: wild-type loci carrying no SNP and knock-in of the various SNPs into the respective loci. The constructs were generated via Vectorbuilder. Constructs were sequenced to confirm the expected genotype and to ensure no off-target mutations were introduced. Dual luciferase reporter assays were performed as described previously with minor modifications^11,58. Briefly, GFP, POLR2A, NRF1, CTCF, FOSL1, GATA1, GATA2, CEBPB, or NFE2 overexpressing HEK293 cells or K562 cells were co-transfected with one of the two pGL3 luciferase vectors described above as well pGL3 control according to the manufacturer’s instructions. Firefly and Renilla luciferase reporter activity of cell extracts were measured using the Dual-Glo Luciferase Assay System (Promega) on a microplate reader according to the manufacturer’s instructions. Each treatment was performed in duplicate and the experiment was repeated three times. Assay primer information is given in Supplementary Table ¹².