Inexpensive protocol for single-cell X-chromosome inactivation analysis: Xist expression and H3K27me3 staining

Abstract

The X-chromosome status is an important marker of mammalian cell embryonic differentiation. Both female X-chromosomes are transcriptionally active (Xa) at the blastocyst stage, then one of the X-chromosomes undergoes random inactivation (Xi). Naïve pluripotent stem cells have both Xa, while primed pluripotent stem cells and differentiated cells – XaXi. When pluripotent stem cells are generated from non-model species, it is important to analyze X-chromosome status, as well as pluripotency marker gene expression at the early stages, to minimize time and cost. Thus, the X-chromosome state may point to the pluripotency status of the newly derived pluripotent cells, and early analysis of pluripotency markers would facilitate the cell selection. We present two detailed protocols of X-chromosome inactivation analysis: a single-cell analysis based on cell lysis with subsequent reverse transcription PCR, and immunofluorescent H3K27me3 staining. The H3K27me3 staining marks transcriptionally inactive chromatin. The single-cell lysis for RNA isolation is performed in a small volume of PCR-compatible non-ionic detergents, Nonidet P-40 and Tween-20, with BSA. Subsequent cDNA synthesis is performed in the same tube. This method could be used for Xist amplification, as an early X-chromosome inactivation marker, as well as for other genes, such as Rex1, expressed in mammalian pluripotent cells. We have successfully applied those protocols for X-inactivation analysis in the American mink embryonic fibroblasts and three lines of induced pluripotent stem cells. Thus, the proposed protocols allow fast and inexpensive testing of pluripotency and X-inactivation markers on a single-cell level.

Keywords

• single cells
• neovison vison
• american mink
• xist
• rex1
• hk1
• h3k27me3