Project Summary/Abstract How the identical genome sequence produces diverse cell types during development remains a fundamental question in biology. Recent technology advancements in single-cell genomics provided excellent opportunities to study the molecular profiles during development and in disease at unprecedented resolution. However, monitoring individual modalities from single cells at a time runs the risk of obtaining only partial pictures from the complex regulatory network. Multi-modal single-cell genomics tools would be desired to overcome this limitation. I recently invented a method for ultra-high-throughput joint analysis of open chromatin and transcriptome from the same single cells (Paired-seq) and demonstrated its potential for comprehensive investigations of the cell- type-specific regulatory programs from heterogenous brain tissues. In this K99/R00 application, I propose to further develop a set of new single-cell multi-omics tools to study the dynamic and cell-type-specific regulatory circuits during mammalian development. I will improve the sensitivities and coverages of Paired-seq and develop a computational method for single-cell multi-omics analysis from the phenotypic level (Aim1). Subsequently, I will further develop a method for high-throughput single-cell joint analysis of histone modifications/transcription factors binding with gene expression (Paired-tag) for analysis of molecular programs from the mechanistic level (Aim2). Finally, I will apply these technologies to study the dynamic and cell-type-specific molecular programs in mammalian developing germ cells, and to identify and validate novel regulators during this process (Aim3). Overall, the results from this proposal will provide new technologies for the study of epigenetic programs in complex tissues and during development at single-cell resolution, and providing more complete views of the gene regulatory circuits during mammalian germ cell development. My career goal is to lead an independent research group focusing on integrating novel experimental and computational technologies to understand the underlying principles controlling mammalian development. During the K99 phase, I will continue to receive experimental and computational training from my postdoctoral mentor Dr. Ren and collaborators/advisory committee at UC San Diego and the Salk Institute. The rigorous mentored support and results obtained in the K99 phase will facilitate my transition to an independent investigator in the R00 phase and lay the foundation for my future career.