High-Throughput Protein Production Combined with High- Throughput SELEX Identifies an Extensive Atlas of Ciona robusta Transcription Factor DNA-Binding Specificities

Transcription factors (TFs) control gene transcription, binding to specific DNA motifs located in cis-regulatory elements across the genome. The identification of TF-binding motifs is thus an important aspect to understand the role of TFs in gene regulation. SELEX, Systematic Evolution of Ligands by EXponential enrichment, is an efficient in vitro method, which can be used to determine the DNA-binding specificity of TFs. Thanks to the development of high-throughput (HT) DNA cloning system and protein production technology, the classical SELEX assay has be extended to high-throughput scale (HT-SELEX).We report here the detailed protocol for the cloning, production, and purification of 420 Ciona robusta DNA BD. 263 Ciona robusta TF DNA-binding domain proteins were purified in milligram quantities and analyzed by HT-SELEX. The identification of 139 recognition sequences generates an atlas of protein-DNA-binding specificities that is crucial for the understanding of the gene regulatory network (GRN) of Ciona robusta. Overall, our analysis suggests that the Ciona robusta repertoire of sequence-specific transcription factors comprises less than 500 genes. The protocols for high-throughput protein production and HT-SELEX described in this article for the study of Ciona robusta TF DNA-binding specificity are generic and have been successfully applied to a wide range of TFs from other species, including human, mouse, and Drosophila.