Polycomb Underlies Transcriptional Heterogeneity in Lineage Priming of Embryonic Stem Cells [gene expression]

Quantitation of polyA mRNA levels in subpopulations of the HexVenus reporter (clone HV5.1) mouse ES cell line growing under self-renewing conditions. Subpopulations were identified and isolated based on the expression of the ES cell surface marker SSEA 1 and the expression of the venus protein. At approximately 70% confluence, cells were trypsinised, resuspended in FACs buffer (10%FCS in PBS) and incubated with a mouse monoclonal antibody to SSEA 1 (MC-480, Developmental Hybridoma Studies Bank, University of Iowa). Cells were then incubated with an Alexa-647 conjugated anti-mouse IgM antibody (Invitrogen) and subpopulations were fractionated by flow cytometry. The aim was to identify genes which are differentially expressed between FACS-sorted HV-S+ and HV+S+ primed mESC populations. mESCs carrying a fluorescently tagged Hhex-Venus (HV) reporter construct were FACS sorted into Ssea1+HV- and Ssea1+HV+ populations. RNA was isolated from these populations immediately following FACS sorting and hybridised to Agilent 44k expression arrays. This process was performed as two independent biological replicates.