Expression profiling by high throughput sequencing Genome binding/occupancy profiling by high throughput sequencing
Summary
A systematic interrogation of male germ cells is key to complete understanding of molecular mechanisms governing spermatogenesis and the development of new strategies for infertility therapies and male contraception. Here we develop an approach to purify all types of homogeneous spermatogenic cells by combining transgenic labelling and synchronization of the cycle of the seminiferous epithelium, and subsequent single-cell RNA-sequencing. We reveal extensive and previously uncharacterized dynamic processes and molecular signatures in gene expression, as well as specific patterns of alternative splicing, and novel regulators for specific stages of male germ cell development. Our transcriptomics analyses led us to discover discriminative markers for isolating specific stages of round spermatids, and identify different embryo developmental potential between early and late stage spermatids, providing evidence that maturation of round spermatids impacts on embryo development. This work provides valuable insights into mammalian spermatogenesis, and a comprehensive resource for the complete elucidation of gametogenesis.
Overall design
2-dpp mice with Lin28-YFP and Vasa-dTomato were pipette fed WIN 18,446 (MP) for 7 consecutive days. At Day 8 of WIN 18,446 treatments, these animals were received an injection of RA (Sigma), and then left to recover. At the right timepoint,we isolate the Vasa-dTomato positive synchronous spermatogenic cells to perform single-cell RNA-seq. For functional study, we performed Sox30 ChIP-seq.
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