Mapping the chromatin accessibility landscape of zebrafish embryogenesis at single-cell resolution by SPATAC-seq

Zebrafish is a popular model for studies of embryonic development. Recent surveys of zebrafish embryogenesis with single-cell transcriptomics have uncovered hundreds of cell types, but the dynamic accessible regions of the genome that determine regulatory programs responsible for the unique identity and function of each cell type during embryogenesis lack detailed study. Here, we present SPATAC-seq: a split-pool ligation-based high-throughput single-cell assay for transposase-accessible chromatin using sequencing. Using SPATAC-seq, we profiled chromatin accessibility in more than 808,046 individual nuclei across 20 developmental stages spanning the sphere stage to the early larval protruding mouth stages in a single experiment. Using this chromatin accessibility map, we identified 604 cell types and inferred their developmental relationships. We also identified 959,040 candidate cis-regulatory elements (cCREs) and delineate development-specific cCREs, as well as transcription factors defining diverse cell identities. Importantly, transient reporter assays confirmed that a majority of tested cCREs exhibited robust enhanced EGFP expression in restricted cell types or tissues. Finally, we explored gene regulatory programs that drive pigment and notochord cell differentiation. Our work provides a comprehensive and valuable open resource for exploring transcriptional regulators that determine cell fate specification in zebrafish embryogenesis. Wild-type zebrafish strain Tübingen (TU) line were maintained in standard zebrafish units at Core Facilities, school of life sciences, Tsinghua University. Naturally spawned fish embryos were collected at the 1-cell stage and the time of fertilization was used to stage each clutch. Embryos were maintained in Holtfreter’s solution at 28.5 °C for time course experiments and processed for bulk ATAC-seq and SPATAC-seq at the indicated times. Embryo nuclei of 20 developmental stages were extracted via previously described methods (Corces MR, et al. Nat Methods. 2017; Cao J, et al. Nature. 2019). The isolated nuclei were used for bulk ATAC-seq with TruePrep DNA Library Prep Kit V2 for Illumina (TD501, Vazyme), SPATAC-seq and 10x Genomics scATAC-seq (See more detailed method procedures from our paper). SPATAC-seq and bulk ATAC-seq libraries were sequenced with paired-end 150-bp reads on Illumina NovaSeq 6000 platform or DNBSEQ-T7. 10x Genomics scATAC-seq were sequenced with paired-end 100-bp reads on MGISEQ-2000RS, and i5 16 bp were linked at the end of Read 2. Please note that the Series supplementary *bw files were genearated from all 535 samples together.