From the motif analysis, ERVKs with open chromatin state in hTSCs are enriched for TSC-related transcription factor motifs such as and in the na?ve mESCs drove the cells towards a TSC-like cell fate, but not mEpiSCs . Early studies characterizing hESC-derived trophoblast-like cells focused on human chorionic gonadotropin production and cellular invasion capacity. to differentiate and give rise to the whole organism has fascinated biologists for decades. Epigenetic regulation, including histone modifications, histone variant substitutions, maternal factors, DNA methylation, and imprinting, plays a crucial role in the specification and determination of cell fate. Epigenetic factors can change chromosome conformation and the weak interacting forces , leading to differential gene expression across cell types. Molecular biology techniques such as fluorescence microscopy and RNA interference have only answered particular aspects of the underlying mechanisms. However, more delicate approaches are required to solve increasingly sophisticated questions in the field. The discoveries of a totipotent subpopulation within mouse embryonic stem cell (mESCs) culture , expanded potential stem cells (EPSC) [3, 4], and induced pluripotent stem cells with higher potency  have reignited the interest in developing media that are capable of maintaining cells with increased differentiation potential. Studies suggest that such potential is linked to the bivalent chromatin [6, 7] and depletion of inhibitory markers that stabilise the cell fate . The mESCs and primed human ESC (hESCs) are capable of Idasanutlin (RG7388) differentiating into the trophoblast lineage upon manipulation [9, 10]. However, it remains unknown whether the transdifferentiation into the trophoblast lineage happens after the transition to the totipotent state  or induced directly from Idasanutlin (RG7388) the alternate pluripotent state . Recent developments in single-cell technology have allowed us to look deeper into cellular networks involving chromatin state and epigenetic regulators in early embryogenesis [13C15]. These proof of concept studies have showcased the potential of single-cell technology in meeting Keratin 5 antibody the needs of the field. 2. Single-Cell and Low-Input Techniques Cellular heterogeneity primes cells towards different lineages and is difficult to study in the context of the embryogenesis. Traditional methods employing the expression of fluorescent proteins and observational studies by perturbing critical factors that are known to be involved in the formation of embryos are both time consuming and inefficient. Additionally, certain cell types with smaller population sizes are easily masked in the bulk analysis. Ever since the advent of single-cell technology in 2009 2009 , which permitted the analysis of the mouse embryonic transcriptome, the field has quickly adapted this concept to questions highly relevant to epigenetic regulation. However, these methods remain technically challenging, especially during the process of amplifying Idasanutlin (RG7388) signals from each cell while suppressing unspecific noises. Epigenetic studies often involve a bulk analysis of materials pooled together using millions of cells to derive the most accurate map, which is not practical in studies involving early embryos. To this end, various groups have employed different methods, such as multiple rounds of bar coding and specialised beads to improve capturing and accuracy of amplification of the epigenome [14, 17, 18] (Figure 1). Open in a separate window Figure 1 Summary of the comparison of different single-cell and low-input techniques to assess chromatin Idasanutlin (RG7388) structure [16C23, 27C31, 33, 34, 36C38]. Created with http://BioRender.com/. Chromatin accessibility reflects, to some degree, the expression status of genes by controlling the exposure of genomic regions to transcription factors (TFs) and other DNA-binding elements. There are currently four approaches to analyse chromatin accessibility in a single cell. Three of them quantify enrichment of DNA fragments after enzymatic DNA cleavage of accessible regions. The assay for transposase-accessible chromatin using sequencing (ATAC-seq) employs the hyperactive transposase Tn5 which simultaneously cleaves Idasanutlin (RG7388) and inserts itself to the accessible regions and ligates sequencing indexes containing adaptors to these regions in each cell (Figure 1). The resultant DNA fragments are amplified polymerase chain reaction (PCR), and short fragments are selected to remove partially digested fragments that are longer in length [19C21]. A second approach employs the so-called DNase I hypersensitive site sequencing (DNase-seq), whereby DNase-sensitive chromatin is cleaved and further processed with.