EXPERIMENT_TITLE: GSM6597328: Hi-C naive; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: Hi-C 2.0 was performed as described previously (Belaghzal et al., 2017). Briefly, 10 cm plates of naïve and primed hESCs were crosslinked and aliquoted into 5.5×106 cell aliquots. Each aliquot was separately treated as a Hi-C sample. Each sample underwent plasma membrane lysis and removal of the cytoplasm before nuclear semi-permeabilization. Next, semi-permeable crosslinked nuclei were digested with the DpnII restriction enzyme. The sticky ends were filled in with dNTPs and biotinylated dCTP before blunt-end ligation. After ligation, cross-linking was reversed, nuclei were broken down, and correctly sized biotinylated DNA was purified out of the larger DNA pool. Illumina sequencing primers were added to these biotinylated fragments, which were subsequently amplified.
STUDY_DESCRIPTOR_STUDY_ABSTRACT: In naïve human pluripotent stem cells (hPSCs) that represent the pre-implantation embryonic states, epigenetic regulators for cell identity remain poorly defined.  One of the major remaining questions in naïve stem cell biology is how female cells mediate and regulate X chromosome inactivation (XCI). Recent studies incorporating single-cell RNA sequencing (scRNA-seq) have demonstrated that transcript-based technologies are insufficient to unequivocally resolve XCI regulation in a human system. Instead, 3D genomics shows immense promise for studying XCI by interrogating changes to the X chromosomes' 3D states. Here, we sought to characterize the 3D state of the X chromosome in naïve and primed hPSCs. Using chromatin tracing, we analyzed the folding conformations of whole X chromosomes with megabase genomic resolution in multiple naïve and primed hPSC lines. Our data found that X chromosomes in female naive hPSCs exhibit a range of spatial folding conformations similar to the active X chromosome (Xa) and the inactive X chromosome (Xi) in somatic cells; these data suggest that naïve hPSCs have initiated XCI. As XCI process ultimately generates detectable differences in volume between the Xa and the Xi, we measured the radius of gyration of individual X chromosome copies across various hPSC cell lines and culture conditions. In naïve hPSCs, our results show that the X chromosomes with Xi-like conformations are not more compact than those with Xa-like conformation. This finding is directly counter to the Xi compaction typically observed in post-XCI female somatic cells. These contradictory 3D signatures of XCI suggest that female naïve hPSCs have initiated the XCI process, but are poised before XCI-driven silencing in a metastable state. As observed in H7 naïve hPSCs, this metastable state can be abolished through classically noted means, such as the loss of Xp chromatin, leading to the deleted p X chromosome (dXp). The Xp loss observed here seems to drive XIST expression and accumulation around the dXp chromosome. Overall, our findings provide insight into the previously undefined X chromosome status in naïve hPSCs in single chromosome resolution, and are critical in understanding the unique epigenetic regulation in early embryonic cells. Overall design: Hi-C for human naive and primed hESCs
SAMPLE_TITLE: Hi-C naive
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Pool_Member_@attributes_sample_title: Hi-C naive
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