RESUMO
Three-dimensional (3D) chromosome organization is a major factor in genome regulation and cell-type specification. For example, cis-regulatory elements, known as enhancers, are thought to regulate the activity of distal promoters via interaction in 3D space. Genome-wide chromosome conformation capture (3C)-technologies, such as Hi-C, have transformed our understanding of how genomes are organized in cells. The current understanding of 3D genome organization is limited by the resolution with which the topological organization of chromosomes in 3D space can be resolved. Micro-C-XL measures chromosome folding with resolution at the level of the nucleosome, the basic unit of chromatin, by utilizing micrococcal nuclease (MNase) to fragment genomes during the chromosome conformation capture protocol. This results in an improved signal-to-noise ratio in the measurements, thus facilitating the better detection of insulation sites and chromosome loops compared to other genome-wide 3D technologies. A visually supported, detailed, step-by-step protocol for preparing high-quality Micro-C-XL samples from mammalian cells is presented in this article.
Assuntos
Cromatina , Nucleossomos , Animais , Cromatina/genética , Mamíferos , Nuclease do Micrococo , Regiões Promotoras GenéticasRESUMO
Several distinct differentiation protocols for deriving pancreatic progenitors (PPs) from human pluripotent stem cells have been described, but it remains to be shown how similar the PPs are across protocols and how well they resemble their in vivo counterparts. Here, we evaluated three differentiation protocols, performed RNA and assay for transposase-accessible chromatin using sequencing on isolated PPs derived with these, and compared them with fetal human pancreas populations. This enabled us to define a shared transcriptional and epigenomic signature of the PPs, including several genes not previously implicated in pancreas development. Furthermore, we identified a significant and previously unappreciated cross-protocol variation of the PPs through multi-omics analysis and demonstrate how such information can be applied to refine differentiation protocols for derivation of insulin-producing beta-like cells. Together, our study highlights the importance of a detailed characterization of defined cell populations derived from distinct differentiation protocols and provides a valuable resource for exploring human pancreatic development.