Interactions between transcription factors and regulatory DNA can be described by gene regulatory networks. These networks provide a systems-level view of embryonic tissue development. Here, we describe a protocol for the isolation, identification, and experimental manipulation of tissue-specific cis-regulatory elements during zebrafish embryonic development using low-input ATAC-seq. With the methods described, genome-wide assessments of regulatory DNA in small populations of developing tissues can be identified, allowing for the construction of gene regulatory networks.