Project description:Single-cell polyadenylation site quantification 48 single cells each of murine embryonic stem cells maintained in FCS+LIF medium ("ESC"), embryonic stem cells maintained in serum free, 2i containing medium ("2i") and neural stem cells ("NSC") were sequenced by BATSeq, a single cell transcriptomic protocol for mapping polyadenylation sites in single cells.
Project description:Naive pluripotent embryonic stem cells (ESCs) and embryonic germ cells (EGCs) are derived from the preimplantation epiblast and primordial germ cells (PGCs), respectively. We investigated whether differences exist between ESCs and EGCs, in view of their distinct developmental origins. PGCs are programmed to undergo global DNA demethylation; however, we find that EGCs and ESCs exhibit equivalent global DNA methylation levels. Importantly, inhibition of Erk and Gsk3b by 2i conditions leads to pronounced reduction in DNA methylation in both cell types. This is driven by Prdm14 and is associated with downregulation of Dnmt3a and Dnmt3b. However, genomic imprints are maintained in 2i, and we report derivation of EGCs with intact genomic imprints. Collectively, our findings establish that culture in 2i instills a naive pluripotent state with a distinctive epigenetic configuration that parallels molecular features observed in both the preimplantation epiblast and nascent PGCs. EGC lines were derived from E8.5 mouse embryos using three different protocols: FCS+LIF on MEFs (FCS, n = 4)), FCS+LIF on MEFs for 48 hours followed by 2i+LIF (2is, n = 4), and direct derivation into 2i+LIF (2i, n = 4). ESC lines were derived in either FCS+LIF on MEFs or in 2i+LIF conditions and once established the cell lines were also switched between the two culture environments (n = 5 for each culture condition). All cell lines were derived from genetically identical embryos.