GSE137693
Integrating Enhancer RNA Signatures with Diverse Omics Data identifies characteristics of transcription initiation in pancreatic islets
Identifying active regulatory elements and their characteristics is critical to understand gene regulatory mechanisms and subsequently better delineating biological mechanisms of complex diseases and traits. Studies have shown that active enhancers can be transcribed into enhancer RNA (eRNA). We identified actively transcribed  elements in human pancreatic islets by performing cap analysis of gene expression (CAGE) across 70 islet samples. We used the self-transcribing active regulatory region sequencing (STARR-seq) assay in a rat pancreatic islet beta cell line to experimentally validate the regulatory activity of the CAGE-identified transcribed elements. Overall design: We selected a total of 7,188 islet CAGE elements (198bp each) to test in the STARR-seq assay. We generated 230-bp synthetic oligos (198bp CAGE element flanked by 16bp anchor sequences, Agilent) and cloned into the STARR-seq backbone plasmid. We inserted 16-bp random nucleotides ('barcodes') in the plasmid library using Gibson assembly. We electroporated 10 ug of barcoded STARR-seq library into 25 million the 832/13 rat insulinoma cell line for each of the three biological replicates, and harvested the cells twenty-fours later. We isolated total RNA and purified mRNA using oligo(dT) beads, treated with RNase-free DNaseI and then reverse transcribed it into cDNA. We performed PCR amplification and sequenced the libraries on the Illumina HiSeq platform.