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| Status |
Public on Nov 20, 2023 |
| Title |
HaCaT_Tet-on-Luc, DoxD3, CTCF HiChIP |
| Sample type |
SRA |
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| Source name |
HaCaT
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HaCaT
cell type: Keratinocyte
genotype: Tet-on-Luc (pTet-on-Advanced, pTRE-Tight-Luc)
treatment: Treated with 0.1μg/ml Dox for 3 days
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| Treatment protocol |
Doxycycline (Dox) was added into the culture medium to a final concentration of 0.1μg/ml to induce the expression of IKAROS (HaCaT_Tet-on-Ikzf1) or Luciferase (HaCaT_Tet-on-Luc).
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| Growth protocol |
The HaCaT keratinocyte cells were grown at 37 °C in DMEM medium supplemented with 10% ‘Tet System Approved FBS’ (Clontech), penicillin (100 units/ml) and streptomycin sulfate (100 μg/ml) in humidified atmosphere of 5% CO2.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
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.
HiChIP assays were performed according to the previously published protocol (Mumbach et al., 2016) with minor modifications. Briefly, ~10 million cells were crosslinked with 1% Formaldehyde for 10 mins at room temperature and lysed with Hi-C lysis buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 0.2% NP40) supplemented with protease inhibitors for 30 mins at 4C. Chromatin was digested by the DpnII restriction enzyme for two hours at 37C. The 5’ overhang of the digested DNA were filled with dCTP, dGTP, dTTP and biotinl14-dATP by DNA Polymerase I, Large Klenow Fragment, and then ligated by T4 DNA ligase for 4 hours at room temperature. Chromatin was sonicated in Nuclear Lysis buffer (50 mM Tris-HCl pH 7.5, 10 mM EDTA, 0.6% SDS), 1:2 diluted in ChIP Dilution buffer (2% Triton X-100, 300 mM NaCl, 0.2% NaDoc), and then processed with regular chromatin immunoprecipitation using antibodies against H3K27ac, SMC1a, CTCF, or IKAROS. The DNA junctions labelled by Biotin-14-dATP were then enriched by Streptavidin C1 magnetic beads and processed with Nextera DNA library preparation (Illumina). The DNA libraries were size-selected by Ampure XP beads (300-700bp) and sequenced in house with an Illumina NEXTseq550 system and a Novaseq 6000 system at the Knapp Center for Biomedical Discovery, University of Chicago.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
instrument model: Illumina NEXTseq550 and Novaseq6000
Sequencing reads were aligned to the human hg19 assembly and processed into normalized contact maps with the HiC-Pro 2.8.0 pipeline (Servant et al., 2015) on the DNAnexus platform.
Default settings were used to remove duplicate reads, assign reads to restriction fragments, filter for valid pairs, and generate raw and ICE normalized interaction matrices at a range of resolutions.
For visualization by Juicebox (v1.11.08) valid pairs were converted to .hic files using the script “hicpro2juicebox.sh”.
Assembly: hg19
Supplementary files format and content: hic
Library strategy: HiChIP
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| Submission date |
May 07, 2023 |
| Last update date |
Nov 20, 2023 |
| Contact name |
Katia Georgopoulos |
| E-mail(s) |
katia.georgopoulos@cbrc2.mgh.harvard.edu
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| Phone |
617-7264445
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| Organization name |
Harvard Medical School
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| Department |
CBRC
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| Lab |
Georgopoulos
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| Street address |
1st and 13th St
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| City |
Charlestown |
| State/province |
MA |
| ZIP/Postal code |
02129 |
| Country |
USA |
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| Platform ID |
GPL24676 |
| Series (2) |
| GSE231881 |
A multiscale 3D chromatin architecture that controls development of the humoral immune system is assembled by IKAROS [HiChIP_HaCaT] |
| GSE232490 |
A multiscale 3D chromatin architecture that controls development of the humoral immune system is assembled by IKAROS |
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| Relations |
| BioSample |
SAMN34993216 |
| SRA |
SRX20249101 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7305509_HiChIP_CTCF_HaCaT_Luc_Dox_D3.hic |
413.7 Mb |
(ftp)(http) |
HIC |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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