Abstract：
Over the last decade, single-cell approaches have become the gold standard for studying gene expression dynamics, cell heterogeneity, and cell states within samples. Before single-cell advances, the feasibility of capturing the dynamic cellular landscape and rapid cell transitions during early development was limited. In this paper, a robust pipeline was designed to perform single-cell and nuclei analysis on mouse embryos from embryonic day E6.5 to E8, corresponding to the onset and completion of gastrulation. Gastrulation is a fundamental process during development that establishes the three germinal layers: mesoderm, ectoderm, and endoderm, which are essential for organogenesis. Extensive literature is available on single-cell omics applied to wild-type perigastrulating embryos. However, single-cell analysis of mutant embryos is still scarce and often limited to FACS-sorted populations. This is partially due to the technical constraints associated with the need for genotyping, timed pregnancies, the count of embryos with desired genotypes per pregnancy, and the number of cells per embryo at these stages. Here, a methodology is presented designed to overcome these limitations. This method establishes breeding and timed pregnancy guidelines to achieve a higher chance of synchronized pregnancies with desired genotypes. Optimization steps in the embryo isolation process coupled with a same-day genotyping protocol (3 h) allow for microdroplet-based single-cell to be performed on the same day, ensuring the high viability of cells and robust results. This method further includes guidelines for optimal nuclei isolations from embryos. Thus, these approaches increase the feasibility of single-cell approaches of mutant embryos at the gastrulation stage. We anticipate that this method will facilitate the analysis of how mutations shape the cellular landscape of the gastrula.
摘要：
在过去的十年里，单细胞方法已成为研究基因表达动力学的金标准，细胞异质性，和样品中的细胞状态。在单细胞进步之前，在早期发育过程中捕获动态细胞景观和快速细胞过渡的可行性是有限的。在本文中,我们设计了一个稳健的流程,对胚胎期E6.5~E8天的小鼠胚胎进行单细胞和细胞核分析,对应于胃泌素的开始和完成.产气是发育过程中建立三个胚层的基本过程：中胚层，外胚层,和内胚层，对器官发生至关重要。关于应用于野生型围胃胚胎的单细胞组学，有大量文献。然而,突变胚胎的单细胞分析仍然很少，并且通常仅限于FACS分类的种群。这部分是由于与基因分型需求相关的技术限制，定时怀孕,每次怀孕所需基因型的胚胎计数，每个胚胎在这些阶段的细胞数量。这里,提出了一种旨在克服这些限制的方法。该方法建立了育种和定时妊娠指南，以实现具有所需基因型的同步妊娠的更高机会。胚胎分离过程中的优化步骤与同一天的基因分型方案（3小时）相结合，允许在同一天进行基于微滴的单细胞，确保细胞的高活力和稳健的结果。该方法还包括从胚胎中最佳核分离的指南。因此，这些方法增加了单细胞方法在原肠胚形成阶段突变胚胎的可行性.我们预计这种方法将有助于分析突变如何塑造胃细胞的细胞景观。
