Oulhen Nathalie, Foster Stephany, Wray Greg, Wessel Gary
Department of Molecular and Cell Biology and Biochemistry, Brown University, Providence, RI, United States.
Department of Biology, Duke University, Durham, NC, United States.
Methods Cell Biol. 2019;151:127-158. doi: 10.1016/bs.mcb.2018.11.018. Epub 2019 Jan 2.
Gene regulatory networks reveal how transcription factors contribute to a dynamic cascade of cellular information processing. Recent advances in technologies have enhanced the toolkit for testing GRN mechanisms and connections. Here we emphasize three approaches that we have found important for interrogating transcriptional mechanisms in echinoderms: single cell mRNA sequencing (drop-seq), nascent RNA detection and identification, and chromatin immunoprecipitation (ChIP). We present these applications in order since it is a logical experimental protocol. With preliminary information from bulk mRNA transcriptome analysis and differential gene expression studies (DE-seq), one may need to test in what specific cells important genes may be expressed and to use single cell sequencing to define such links. Nascent RNA analysis with the Click-iT chemistry allows the investigator to deduce when the RNA was transcribed, not just identify its presence, and ChIP allows the investigator to study direct interactions of putative transcriptional regulators with the gene promoter of interest. This flow of thinking, and the technologies to support it, is presented here for echinoderms. While many of the procedures are general and applicable to many organisms and cell types, we emphasize unique aspects of the protocols for consideration in using echinoderm embryos, larvae, and adult tissues.
基因调控网络揭示了转录因子如何促成细胞信息处理的动态级联反应。技术上的最新进展增强了用于测试基因调控网络机制和联系的工具集。在此，我们着重介绍三种我们发现对探究棘皮动物转录机制很重要的方法：单细胞mRNA测序（drop-seq）、新生RNA检测与鉴定以及染色质免疫沉淀（ChIP）。我们按顺序介绍这些应用，因为这是一个符合逻辑的实验方案。借助来自大量mRNA转录组分析和差异基因表达研究（DE-seq）的初步信息，可能需要测试重要基因可能在哪些特定细胞中表达，并使用单细胞测序来确定此类联系。使用Click-iT化学方法进行新生RNA分析可使研究人员推断RNA何时被转录，而不仅仅是确定其存在，ChIP则使研究人员能够研究假定的转录调节因子与感兴趣基因启动子的直接相互作用。这里针对棘皮动物展示了这种思维流程及其支持技术。虽然许多程序是通用的，适用于许多生物体和细胞类型，但我们强调了在使用棘皮动物胚胎、幼虫和成年组织时需考虑的方案独特之处。
