Abstract
Cells are the complex product of gene expression programs that involve the coordinated transcription of thousands of genes controlled by cis-regulatory elements (cis-REs). Therefore, identification of cis-REs is the key to decipher the mechanisms underlying the regulation of gene expression. Here, we describe a simple and time-effective protocol of fine-mapping cis-REs by using an electrophoresis mobility shift assay (EMSA)-based, in vitro, high-throughput (HTP) technique called regulatory element-sequencing (Reel-seq). Reel-seq can be applied to identify cis-REs at a high resolution and sensitivity over large genome regions, in a systematic and continuous manner. It can be used to prioritize candidate cis-REs as a complement to the existing approaches, such as massive parallel reporter assay (MPRA), chromatin immunoprecipitation DNA-sequencing (ChIP-seq), and the assay for transposase-accessible chromatin sequencing (ATAC-seq). Graphical : Generation of the Reel-seq Library 1 and 2 (A) and identification of cis-REs by an electrophoresis mobility shift assay (EMSA)-based Reel-seq screen (B). NE: nuclear extract; NGS: next generation sequencing.
Keywords:
EMSA; Gene transcription; High-throughput (HTP) technique; and PCR.; cis-regulating elements (cis-RE).