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| Status |
Public on Dec 20, 2021 |
| Title |
DAXX genome binding/occupancy profiling by high throughput ChIP sequencing |
| Organism |
Homo sapiens |
| Experiment type |
Genome binding/occupancy profiling by high throughput sequencing
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| Summary |
Purpose: The goal of this study is to compare DAXX genome binding/occupancy by high throughput ChIP sequencing in control, wild type DAXX overexpressd cells (WT DAXX OE), and wild type DAXX SUMO interacting motif mutant overexpressd cells (DSM OE).
Methods: The panel of MDA_MB-231 derived cell lines (control, WT DAXX OE, DSM OE) were cultured in complete DMEM. At about 90% confluency, the cells were crosslinked by adding 37 % formaldehyde to the final concentration of 1% for 10 min at room temperature. Crosslinking was stopped by adding glycine to the final concentration of 125 mM. Cells were lifted, washed with cold PBS, and pelleted by centrifugation. The cells were resuspended in a swelling buffer in the presence of the protease inhibitor cocktail (Sigma) and then pelleted and resuspended in the SDS lysis buffer. The lysates were transferred to a Covaris microTUBE and sonicated with an E220 Covaris Ultrasonicator. Chromatin fragmentation (~500 bps) was verified through agarose gel electrophoresis. The fragmented chromatins were diluted and incubated with a control IgG and the DAXX mAb (5G11) along with protein A/G magnetic beads. The beads were washed sequentially with a low salt buffer, high salt buffer, LiCl buffer, and TE buffer (twice). The immunoprecipitated chromatins were eluted at 65 °C for 15 min, and the eluted chromatins were subjected to proteinase K digestion at 65 °C for 3 h. The DNAs were recovered through a Qiagen mini-prep column. The immunoprecipitated DNAs were used for qPCR and library construction and high throughput sequencing using an Illumina Hi-Seq 2500 sequencer.
Results: We surveyed genome-wide occupancy of DAXX using ChIP-seq technology. Overexpression of WT DAXX increased DAXX?s chromatin association specifically with lipogenic pathway genes where DSM OE reverse.
Conclusions: Our study represents the first detailed analysis of DAXX occupancy in lipoegenic gene transciption.
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| Overall design |
Genome sequencing of MDA_MB-231 derived cell lines (control, WT DAXX OE, and DSM OE)
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| Contributor(s) |
Mahmud I, Liao D |
| Citation missing |
Has this study been published? Please login to update or notify GEO. |
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| Submission date |
Dec 13, 2021 |
| Last update date |
Jan 29, 2023 |
| Contact name |
Daiqing Liao |
| E-mail(s) |
dliao@ufl.edu
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| Phone |
(352) 273-8188
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| Organization name |
University of Florida
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| Department |
Anatomy and Cell Biology
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| Lab |
480E
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| Street address |
2033 Mowry Rd
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| City |
Gainesville |
| State/province |
FL |
| ZIP/Postal code |
32610 |
| Country |
USA |
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| Platforms (1) |
| GPL16791 |
Illumina HiSeq 2500 (Homo sapiens) |
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| Samples (3) |
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| Relations |
| BioProject |
PRJNA788463 |
| SRA |
SRP350529 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE190783_ChIPseq_updated.zip |
1.4 Gb |
(ftp)(http) |
ZIP |
| GSE190783_RAW.tar |
173.5 Mb |
(http)(custom) |
TAR (of BED, XLSX) |
| GSE190783_updated_Metadata.xlsx |
21.4 Kb |
(ftp)(http) |
XLSX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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