EXPERIMENT_TITLE: GSM8501047: HEK293, Prm1 positive DAPI high, Hi-C; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: Hi-C libraries were prepared using FLIE Hi-C protocol. Cells were crosslinked with 2% formaldehyde. The cells were lysed in cold lysis buffer (10 mM Tris-HCl, pH 8.0, 10 mM NaCl, 0.2% Igepal CA-630) to permeabilize the nuclei. The nuclei were digested with DpnII restriction enzyme, following by end-filling with biotin-14-dATP, dCTP, dGTP, and dTTP with Klenow. Blunt ends were ligated with T4 DNA ligase, and the formaldehyde crosslinking was reversed by proteinase K (10 mg/ml). DNA was purified using phenol-chloroform and ethanol precipitation, sonicated to an average size of 200 bp, and biotin was removed from non-ligated DNA ends with T4 polymerase. Biotinylated chimeric DNA strands were pulled down using streptavidin beads (Dynabeads C1). Libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina according to the manufacturer's instructions.
STUDY_DESCRIPTOR_STUDY_ABSTRACT: Although the spatial organization of the genome is closely linked to biological function, genome structure is highly stochastic. Within this heterogeneity, specific function-associated structural elements must be maintained, even when the nucleus is deformed due to physiological physical constraints. The radial positioning of genomic loci - their distance from the nuclear periphery - has long been considered an important feature of genome organization which is correlated with both structural elements and genomic activity. In the current study, we developed an experimental system for manipulating the radial positioning of the genome, by expressing the sperm-specific protein Prm1 in somatic cells. By microscopy, we observe that initial Prm1 nuclear foci develop within 72 hours into a large Prm1 focus occupying most of the nuclear interior, while the entire genome is driven towards the nuclear periphery, resulting in a 3-5 fold reduction in the volume that the genome occupies. Noting that this system enables isolation of a pure population of cells with reorganized nuclei, we then used Hi-C to study the effects of this perturbation. Remarkably, we find that interaction patterns are largely robust to this major nuclear reorganization, with minor changes which mostly reflect a strengthening of heterochromatin self-interactions. Our experimental system provides means for manipulating nuclear organization in a reproducible manner, potentially allowing to examine radial positioning decoupled from other features of genome organization. Highlighting the complementary nature of microscopic and genomic methods, our work further suggests a remarkable resilience of genome structure such that large-scale nuclear changes, including chromosome compression and changes in radial positioning, can occur without extensive alteration of functional genome organization. Overall design: HEK293 cells were transfected with Prm1-mCherry. After 72h, the cells were crosslinked and sorted to isolate Prm1 positive and negative populations, and these were subject to Hi-C (FLIE Hi-C)
SAMPLE_TITLE: HEK293, Prm1 positive DAPI high, Hi-C
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "HEK293"}, {"TAG": "cell line", "VALUE": "HEK293"}, {"TAG": "geo_loc_name", "VALUE": "missing"}, {"TAG": "collection_date", "VALUE": "missing"}]
Pool_Member_@attributes_sample_title: HEK293, Prm1 positive DAPI high, Hi-C
RUN_SET_RUN_Pool_Member_@attributes_sample_title: HEK293, Prm1 positive DAPI high, Hi-C
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