EXPERIMENT_TITLE: GSM4079847: U2OS_dCas9_HiC; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: The sisHi-C library generation was performed as described previously (Du et al., 2017). Briefly, cells were fixed with 1% formaldehyde at room temperature (RT) for 10 min. Formaldehyde was quenched with glycine for 10 min at RT. Cells were washed with 1XPBS for two times and then lysed in 50ul lysis buffer (10 mM Tris-HCl pH7.4, 10 mM NaCl, 0.1 mM EDTA, 0.5% NP-40 and proteinase inhibitor) on ice for 50 min. After spinning at 3000 rpm/min in 4°C for 5 min, the supernatant was discarded with a pipette carefully. Chromatin was solubilized in 0.5% SDS and incubated at 62°C for 10 min. SDS was quenched by 10% Triton X-100 at 37°C for 30 min. Then the nuclei were digested with 50 U MboI at 37°C overnight with rotation. MboI was then inactivated at 62°C for 20 minutes. To fill in the biotin to the DNA, dATP, dGTP, dTTP, biotin-14-dCTP and Klenow were added to the solution and the reaction was carried out at 37°C for 1.5 hours with rotation. The fragments were ligated at RT for 6 hours with rotation. This was followed by reversal of crosslink and DNA purification. DNA was sheared to 300-500 bp with Covaris M220. The biotin-labeled DNA was then pulled down with 10ul Dynabeads MyOne Streptavidin C1 (Life Technology). Sequencing library preparation was performed on beads, including end-repair, dATP tailing and adaptor-ligation. DNA was eluted twice by adding 20ul water to the tube and incubation at 66°C for 20 minutes. 9-15 cycles of PCR amplification were performed with Extaq (Takara). Finally, size selection was done with AMPure XP beads and fragments ranging from 200bp to 1000bp were selected.
STUDY_DESCRIPTOR_STUDY_ABSTRACT: Spatiotemporal control of chromatin structure and dynamics at sub-kilobase to megabase scales is essential for genome function. Epigenetic modification plays important roles in transcriptional regulation. How histone marks regulate genome organization at different scales remain unclear. Here we introduce an EpiGo (Epigenetic perturbation induced Genome organization) system to modify hundreds of genomic loci with H3K9me3 spanning megabases on human chromosome 19 and simultaneously track their changes of genome organization. EpiGo-mediated H3K9me3 spreads more extensively in transcriptionally inactive regions than active regions. H3K9 trimethylation of active regions trigger local chromatin compaction without substantial gene silencing. H3K9 trimethylated loci or regions associate with HP1a condensates and adjacent loci with H3K9me3 coalesce over time. H3K9 trimethylation of inactive regions reshape local genome compartmentalization. These results reveal that H3K9me3 mediates local chromatin compaction or genome compartmentalization upon distinct transcriptional states. Overall design: We introduce an EpiGo (Epigenetic perturbation induced Genome organization) system to modify hundreds of genomic loci with H3K9me3 spanning megabases on human chromosome 19 and simultaneously track their changes of genome organization.
SAMPLE_TITLE: U2OS_dCas9_HiC
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "U2OS cell line"}, {"TAG": "tissue", "VALUE": "human bone osteosarcoma epithelial cell line (female)"}, {"TAG": "genotype/variation", "VALUE": "transduced with PCP-GFP, sgRNA targeting to C19Q, dCas9"}]
Pool_Member_@attributes_sample_title: U2OS_dCas9_HiC
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