Dissecting Transcriptional Heterogeneity in Pluripotency: Single Cell Analysis of Mouse Embryonic Stem Cells.
Mouse Embryonic Stem cells (mESCs) show heterogeneous and dynamic expression of important pluripotency regulatory factors. Single-cell analysis has revealed the existence of cell-to-cell variability in the expression of individual genes in mESCs. Understanding how these heterogeneities are regulated and what their functional consequences are is crucial to obtain a more comprehensive view of the pluripotent state.In this chapter we describe how to analyze transcriptional heterogeneity by monitoring gene expression of Nanog, Oct4, and Sox2, using single-molecule RNA FISH in single mESCs grown in different cell culture medium. We describe in detail all the steps involved in the protocol, from RNA detection to image acquisition and processing, as well as exploratory data analysis.
DHenrique Lab, Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Av. Prof. Egas Moniz, Lisbon, 1649-028, Portugal.|Instituto de Histologia e Biologia do Desenvolvimento, Lisbon, Portugal.
Ana M V
AMV
Guedes
DHenrique Lab, Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Av. Prof. Egas Moniz, Lisbon, 1649-028, Portugal.|Instituto de Histologia e Biologia do Desenvolvimento, Lisbon, Portugal.
Domingos
D
Henrique
DHenrique Lab, Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Av. Prof. Egas Moniz, Lisbon, 1649-028, Portugal. elsa.abranches@gmail.com.|Instituto de Histologia e Biologia do Desenvolvimento, Lisbon, Portugal. elsa.abranches@gmail.com.
Elsa
E
Abranches
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000235:genetics; D020869:Gene Expression Profiling; D018740:Genetic Heterogeneity; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D051379:Mice; D000066450:Mouse Embryonic Stem Cells / Q000166:cytology*; D000071317:Nanog Homeobox Protein / Q000235:genetics; D050814:Octamer Transcription Factor-3 / Q000235:genetics; D039904:Pluripotent Stem Cells / Q000166:cytology; D055748:SOXB1 Transcription Factors / Q000235:genetics; D059010:Single-Cell Analysis / Q000379:methods*; D014158:Transcription, Genetic
D000071317:Nanog Homeobox Protein; C475540:Nanog protein, mouse; D050814:Octamer Transcription Factor-3; C494174:Pou5f1 protein, mouse; D055748:SOXB1 Transcription Factors; C527783:Sox2 protein, mouse
Heterogeneity; Pluripotency; Single-molecule FISH; Stem cells; Stochastic gene expression; Transcription