EXPERIMENT_TITLE: GSM8528458: NIPBL-D7_DMSO_0min_R2_HiC; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: Cells were fixed and processed according to the Arima Genomics HiC+ Kit protocol. For HiC of the RAD21 degron cell lines, cells were fixed and processed as-normal until prior to decrosslinking. At this stage, samples were stained with propidium iodide, and the G1/2N population was sorted in bulk. G1/2N sorted cells then reentered the HiC protocol. Proximity-ligated DNA was first sonicated to ~400 bp using a Covaris M220 with 50 W power, 10% duty factor and 200 cycles per burst for 78 seconds, and then size-selected using AMPure beads. For high-input HiC samples, library preparation was performed using KAPA HyperPrep (Roche #KK8502) according to instructions provided with the Arima Genomics HiC+ Kit, with library preparation done on streptavidin beads. For low-input HiC samples, library preparation was performed using 2S Plus DNA Library Kit (IDT #10009878), with pre-PCR library preparation done in solution before biotin enrichment using Dynabeads MyOne Streptavidin T1 beads, and PCR on bead-bound DNA.
STUDY_DESCRIPTOR_STUDY_ABSTRACT: The organization of the genome in three-dimensional space is highly dynamic, yet how these dynamics are regulated and the role they play in genome function is poorly understood. Here, we utilized acute depletion of NIPBL to characterize the role of cohesin-mediated loop extrusion in vivo. Using this approach, we find that many chromatin loops are rapidly diminished upon loss of NIBPL, consistent with recent single locus imaging studies showing that chromatin loops are transient. However, we also identified a subset of cohesin-dependent chromatin loops that are anchored in repressive chromatin and may be “long-lived”, given that they require NIPBL for their establishment upon mitotic exit, but are persistent when NIPBL is depleted from interphase cells. In addition to the reformation of 3D genome structures, mitotic exit coincides with widespread transcriptional activation. We found that NIPBL is essential for establishing the expression of lineage-defining genes during the M-G1 transition but has diminished impact in the steady-state maintenance of their expression. At genes sensitive to its depletion, NIPBL supports a unique local genome organization defined by greater spatial proximity to nearby enhancers and weaker transcription start site insulation of genomic contacts. Overall, we show that NIPBL-mediated loop extrusion is critical to genome organization and transcriptional regulation in vivo. Overall design: Role of NIPBL in post-mitotic transcriptional reactivation, chromatin association of cohesin, and 3D genome organization.
SAMPLE_TITLE: NIPBL-D7_DMSO_0min_R2_HiC
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "hTERT RPE-1"}, {"TAG": "cell line", "VALUE": "hTERT RPE-1"}, {"TAG": "genotype", "VALUE": "mGreenLantern-P2A-FKBP12(F36V)-NIPBL"}, {"TAG": "clone name", "VALUE": "NIPBL-D7"}, {"TAG": "treatment", "VALUE": "DMSO 120min + 0min"}, {"TAG": "synchronization", "VALUE": "Double thymidine/nocodazole synchronized"}, {"TAG": "geo_loc_name", "VALUE": "missing"}, {"TAG": "collection_date", "VALUE": "missing"}]
Pool_Member_@attributes_sample_title: NIPBL-D7_DMSO_0min_R2_HiC
RUN_SET_RUN_Pool_Member_@attributes_sample_title: NIPBL-D7_DMSO_0min_R2_HiC
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