Identifying gene expression from single cells to single genes

Abstract

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.

Keywords: Chromatin immunoprecipitation; Click-iT nascent RNA labeling; Drop-seq; Embryo dissociation; Single cell mRNA-seq.