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| Status |
Public on Nov 20, 2023 |
| Title |
Large preB_inducible IkE5 deletion, DMSO control, H3K27ac HiChIP, rep2 |
| Sample type |
SRA |
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| Source name |
large Pre-B cell (CD19+CD43+BP1+)
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| Organism |
Mus musculus |
| Characteristics |
cell type: large Pre-B cell (CD19+CD43+BP1+)
genotype: IkE5 fl/fl, Rosa26-ERT2-Cre
treatment: Treat with DMSO for 3-12 days
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| Treatment protocol |
In vitro deletion of IkE5 was induced by addition of of 4-OHT (Sigma H7904, final concentration 0.2 μM ) or with an equal volume of DMSO as control.
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| Growth protocol |
Sorted wild-type IkE5fl/fl Rosa26-ERT2-Cre large pre-B cells were expanded for 6 days on OP9 stroma in DMEM ( D-5671; Sigma) supplemented with 10% FBS (2442; Sigma), 50 μM 2-mercaptoethanol, 100 μg/ml penicillin, 100 μg/ml streptomycin, 1× Glutamax (35050-062; Gibco), 10 mM HEPES (156-30-80; Gibco) and 1× sodium pyruvate (11360-070; Gibco) in the presence of 5ng/ml of IL-7. They were then re-plated onto stromal cells with 0.2 μM of 4-OHT (Sigma H7904) or with an equal volume of DMSO as control. Cells were harvested at different time points (1-18 days) of culture in the presence of 4-OHT or DMSO.
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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 mouse mm10 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: mm10
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 |
GPL24247 |
| Series (2) |
| GSE231880 |
A multiscale 3D chromatin architecture that controls development of the humoral immune system is assembled by IKAROS [HiChIP_LpreB_Inducible] |
| GSE232490 |
A multiscale 3D chromatin architecture that controls development of the humoral immune system is assembled by IKAROS |
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| Relations |
| BioSample |
SAMN34993133 |
| SRA |
SRX20249093 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7305501_HiChIP_H3K27ac_DMSO_R2.hic |
1.2 Gb |
(ftp)(http) |
HIC |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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