Polycomb Underlies Transcriptional Heterogeneity in Lineage Priming of Embryonic Stem Cells [ChIP-Seq and GRO-Seq]. Mus musculus

Chromatin Immunoprecipitation (ChIP) for the histone modification H3K4me3 and H3K27me3 and Genome Wide Run-on seq (GROseq) were performed on subpopulations of the HexVenus reporter mouse ES cell line (clone HV1.5) 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 prior to further processing specific to each method. The aim was to determine if differential gene expression between these populations is primarily mediated at the level of transcription and if these changes are concomitant with changes in chromatin state. Overall design: mESCs carrying a fluorescently tagged Hhex-Venus (HV) reporter construct were FACS sorted into Ssea1+HV- and Ssea1+HV+ populations. Nuclei were prepared from these populations immediately following FACS sorting and then further processed as per the relevant protocol. Two independent biological replicates were prepared for each sample and sequencing libraries were the pooled prior to sequencing or combined bioinformatically following sequencing as outlined for each sample.