It should be noted that the heterozygous SNPs used to identify ASE are markers and not necessarily causal. Therefore, screening multiple variant loci in regulatory regions of those genes is a logical next step. Therefore, we examined the 3′UTR of the genes identified in ASE to test one potential mechanism of gene regulation. We used a high-throughput reporter assay, PASSPORT-seq [13], to screen isolated 3′UTR variants to detect those that lead to gene expression changes. We modified the original protocol to improve the quality of the data. First, we introduced UMIs during the reverse transcription of the mRNA and in the first PCR of the plasmid DNA used as a control to overcome potential PCR biases during the library construction. Second, we added staggered sequences to the PCR primers to reduce problems associated with low sequencing complexity at the beginning of the reads (see Supplementary Materials). A limitation is that we only screened 3′UTR variants; variants in other regulatory regions, such as enhancers and promoters, also play important roles in cis-acting regulation.
