Systematic functional characterization of the non-coding regulatory SNPs associated with central obesity

Genome-wide association studies (GWASs) have identified more than 300 susceptibility loci associated with central obesity. However, the functional understanding of these loci is limited by the fact that most loci are in the non-coding region. To address this issue, in this study we firstly selected 2,034 prioritized SNPs based on fine-mapping and epigenomic annotation analysis. Self-transcribing active regulatory region sequencing (STARR-seq) experiment was then performed to systematically assess the enhancer activity of these prioritized SNPs. Using 2,034 prioritized SNPs, 120-bp DNA sequence centered on each SNP was extracted from the human genomic context, which included reference and alternative alleles. The 15-bp identical adapter sequence was further added on the 5′ and 3′ region for library construction. We synthesized 150-bp long DNA fragments using an oligonucleotides CustomArray(GenScript, USA). Firstly, oligonucleotides were amplified using adapter primers (NEB, USA) and cloned into linearized hSTARR-seq_ORI vector (Addgene, USA) and then we extracted the plasmid library for cell transfection. Secondly, a total of 15μg STARR-seq plasmid library was transfected into 1.0×107 preadipocyte cells. Preadipocytes derived from human subcutaneous adipose tissue was obtained from BLUEFBIO (Shanghai, China). Thirdly, STARR-seq sequencing libraries were prepared and subjected to paired-end sequencing on the Illumina Novaseq 6000 platform. Three biological replicates were performed.