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Summary Here, we describe a highly efficient, medium-throughput strategy for cloning and in vivo screening of putative enhancers using the chick embryo. By incorporating 48 unique nanotags for use in NanoString nCounter® across three different fluorescent reporters and developing a rapid and efficient digestion/ligation type IIs restriction enzyme-based cloning protocol, we develop a multiplexed approach for rapidly identifying enhancer activity. For complete details on the use and execution of this protocol, please see Williams et al. (2019)., Graphical abstract, Highlights • Highly efficient, medium-throughput protocol for cloning putative enhancers • Multiplexed, rapid screening strategy for detecting in vivo enhancer activity • Reproducible, non-mosaic validation of spatial and temporal enhancer activity in vivo • Functional conservation allows cross-species enhancer screening, Here, we describe a highly efficient, medium-throughput strategy for cloning and in vivo screening of putative enhancers using the chick embryo. By incorporating 48 unique nanotags for use in NanoString nCounter® across three different fluorescent reporters and developing a rapid and efficient digestion/ligation type IIs restriction enzyme-based cloning protocol, we develop a multiplexed approach for rapidly identifying enhancer activity.

Summary Chromatin immunoprecipitation with sequencing (ChIP-seq) has been instrumental in understanding transcription factor (TF) binding during gene regulation. ChIP-seq requires specific antibodies against desired TFs, which are not available for numerous species. Here, we describe a tissue-specific biotin ChIP-seq protocol for zebrafish and chicken embryos which utilizes AVI tagging of TFs, permitting their biotinylation by a co-expressed nuclear biotin ligase. Subsequently, biotinylated factors can be precipitated with streptavidin beads, enabling the user to construct TF genome-wide binding landscapes like conventional ChIP-seq methods. For complete details on the use and execution of this protocol, please see Lukoseviciute et al. (2018) and Ling and Sauka-Spengler (2019)., Graphical Abstract, Highlights • Tissue-specific in vivo ChIP for biotinylated DNA-binding proteins of interest • Protocol generates genome-wide binding maps in chicken or zebrafish • ChIP-seq can be performed without antibody usage and cell sorting • Protocol requires a relatively low number of cells as input (100,000–150,000), Chromatin immunoprecipitation with sequencing (ChIP-seq) has been instrumental in understanding transcription factor (TF) binding during gene regulation. ChIP-seq requires specific antibodies against desired TFs, which are not available for numerous species. Here, we describe a tissue-specific biotin ChIP-seq protocol for zebrafish and chicken embryos which utilizes AVI tagging of TFs, permitting their biotinylation by a co-expressed nuclear biotin ligase. Subsequently, biotinylated factors can be precipitated with streptavidin beads, enabling the user to construct TF genome-wide binding landscapes like conventional ChIP-seq methods.