High-throughput functional dissection of noncoding SNPs with biased allelic enhancer activity for insulin resistance-relevant phenotypes

The majority of single nucleotide polymorphisms (SNPs) associated with insulin resistance (IR)-relevant phenotypes by genome-wide association studies (GWASs) are located in noncoding regions, complicating their functional interpretation. Here, we utilized an adapted STARR-seq to systematically detect regulatory activity of 5,987 noncoding GWASs SNPs in three IR-relevant cell lines. We identified 876 SNPs with biased allelic enhancer activity across 133 loci, and further uncovered the genetic regulatory mechanisms underlying functional SNPs through integrating multi-omics analyses. We selected 5,987 noncoding SNPs associated with IR-relevant phenotypes from GWAS catalog and other published studies. A 120-bp genomic sequence centered on each SNP was extracted, with identical adapter sequence added on the 5' and 3' region. Oligonucleotides for 150-bp length SNPs on both reference and alternative alleles were synthesized in a CustomArray. Firstly, STARR-seq plasmid library was constructed using hSTARR-seq_ORI vector and amplified oligonucleotides. Secondly, The plasmid library was transfected into three IR-relevant cell lines (A673, HepG2 and preadipocyte) for 24h. Finally, the plasmid library and RNA extracted after transfection were used to construct input and output sequencing libraries respectively. The library preparations were sequenced on an Illumina Novaseq 6000 platform and paired-end reads were generated. Three biological replicates were performed for each cell line.