EXPERIMENT_TITLE: GSM7305482: HaCaT_Tet-on-Luc, Dox Day3; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: Cells were crosslinked with 1% formaldehyde at RT for 10 minutes, and then quenched by the addition of 0.125 M glycine for 10 minutes. Cells were then washed with ice-cold PBS, snap frozen and stored at -80C before use. In situ Hi-C was performed following the established protocol (Rao et al., 2014) with minor modifications. Briefly, nuclei were permeabilized, and DNA was digested overnight with 100 U DpnII. The ends of the restriction fragments were labeled using biotin-14-dATP and then ligated in a 1.2 mL final volume. After reversal of crosslinks, ligated DNA was purified and sheared to a length of ~400 bp with a Covaris E220 evolution instrument (Covaris, Woburn, MA). Ligation junctions were then pulled down with streptavidin beads, and DNA fragments were end-repaired, dA-tailed and Illumina adapters ligated. Libraries were produced by 8 cycles of PCR amplification with KAPA Hifi DNA polymerase (Roche). The Hi-C libraries were size selected with AMPure XP beads and sequenced in house with an Illumina NEXTseq550 system and a Novaseq 6000 system at the Knapp Center for Biomedical Discovery, University of Chicago.
STUDY_DESCRIPTOR_STUDY_ABSTRACT: A generic level of chromatin organization generated by the interplay between cohesin and CTCF suffices to limit promiscuous interactions between regulatory elements, but a lineage-specific assembly of chromatin that supercedes these constraints is required to configure the genome to support the gene expression changes that guide faithful lineage progression. Using loss-of-function approaches in B cell precursors in vivo we show that IKAROS assembles interactions between sites often separated by megabase distances to configure a significant fraction of the genome in preparation for lymphoid development. Interactions emanating from IKAROS-bound enhancers override CTCF-imposed boundaries and assemble lineage-specific regulatory units built on a backbone of smaller invariant topological domains.In vitro deletion provides temporal resolution to changes in chromatin modifications, loops, and compartmental localization. Gain-of-function experiments in epithelial cells confirm IKAROS' ability to reconfigure chromatin architecture at multiple scales.  While the compaction of the Igk locus required for genome editing represents a function of IKAROS unique to the lymphoid system, the more general function of this lineage-defining DNA binding protein to preconfigure the genome to support lineage specific gene expression and suppress activation of extra-lineage genes provides a paradigm for lineage restriction. Overall design: We induced IKAROS expression in HaCaT cells using a Tet-on strategy and performed in situ Hi-C at different time points to investigate the impact of IKAROS on genome organization in human skin epithelial cells which normally do not express IKAROS.
SAMPLE_TITLE: HaCaT_Tet-on-Luc, Dox Day3
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "HaCaT"}, {"TAG": "cell line", "VALUE": "HaCaT"}, {"TAG": "cell type", "VALUE": "Keratinocyte"}, {"TAG": "genotype", "VALUE": "Tet-on-Luc (pTet-on-Advanced, pTRE-Tight-Luc)"}, {"TAG": "treatment", "VALUE": "Treated with 0.1\u03bcg/ml Dox for 3 days"}, {"TAG": "geo_loc_name", "VALUE": "missing"}, {"TAG": "collection_date", "VALUE": "missing"}]
Pool_Member_@attributes_sample_title: HaCaT_Tet-on-Luc, Dox Day3
RUN_SET_RUN_Pool_Member_@attributes_sample_title: HaCaT_Tet-on-Luc, Dox Day3
RUN_SET_RUN_SRAFiles_SRAFile: [{"Alternatives": [null, null, null], "@attributes": {"cluster": "public", "filename": "SRR24462185.lite", "url": "https://sra-downloadb.be-md.ncbi.nlm.nih.gov/sos3/sra-pub-zq-22/SRR024/24462/SRR24462185/SRR24462185.lite.1", "size": "8657431979", "date": "2023-05-14 07:22:07", "md5": "09bf21b846c69605d5224cffab707a94", "version": "1", "semantic_name": "SRA Lite", "supertype": "Primary ETL", "sratoolkit": "1"}}, {"Alternatives": null, "@attributes": {"cluster": "public", "filename": "SRR24462185", "url": "https://sra-pub-run-odp.s3.amazonaws.com/sra/SRR24462185/SRR24462185", "size": "12681155955", "date": "2023-05-07 22:14:10", "md5": "23c131e1dde4bcf1c84e3965bd3cf99e", "version": "1", "semantic_name": "SRA Normalized", "supertype": "Primary ETL", "sratoolkit": "1"}}, {"Alternatives": null, "@attributes": {"cluster": "public", "filename": "HiC_HaCaT_Luc_Dox_D3_R2.fastq.gz", "size": "11861538108", "date": "2023-05-07 22:07:45", "md5": "27b68ee56cc49a75cb4189299464ce02", "version": "1", "semantic_name": "fastq", "supertype": "Original", "sratoolkit": "0"}}, {"Alternatives": null, "@attributes": {"cluster": "public", "filename": "HiC_HaCaT_Luc_Dox_D3_R1.fastq.gz", "size": "10962475872", "date": "2023-05-07 22:05:46", "md5": "7c2a9255c85c23e15fe11148c99da81d", "version": "1", "semantic_name": "fastq", "supertype": "Original", "sratoolkit": "0"}}]