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| Status |
Public on Oct 31, 2023 |
| Title |
Input_K562dBRD9 |
| Sample type |
SRA |
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| Source name |
hematopoietic cells (K562 cells)
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| Organism |
Homo sapiens |
| Characteristics |
cell line: K562
cell type: hematopoietic, parental cells
treatment: 100nM dBRD9
chip antibody: None
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| Treatment protocol |
K562 parental cells received DMSO and 100nM dBRD9 treatment for 4 days.
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| Growth protocol |
The K562 cell lines were cultured in RPMI with 10% FCS and 1% P/S
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were fixed with 2mM DSG (Thermo Fisher Scientific, 20593) for 30 min at room temperature and replaced with fixing buffer (50 mM HEPES-NaOH (pH 7.5), 100 mM NaCl, 1 mM EDTA) containing 1% paraformaldehyde (Electron Microscopy Sciences, 15714) and crosslinked for 10 min at 37°C.Crosslinking was quenched by adding glycine to a final concentration of 0.125 M. The nuclear fraction was extracted by first resuspending the pellet in 1 ml of lysis buffer (50 mM HEPES-NaOH (pH 8.0), 140 mM NaCl, 1mM EDTA, 10% glycerol, 0.5% NP-40, and 0.25% Triton X-100) for 10 min at 4°C. Cells were pelleted, and washed in 1 ml of wash buffer (10 mM Tris-HCL (pH 8.0), 200 mM NaCl, 1 mM EDTA) for 10 min at 4°C. Cells were then pelleted and resuspended in 1 ml of shearing buffer (10 mM Tris-HCl (pH 8), 1 mM EDTA, 0.1% SDS) and sonicated.Lysate was centrifuged for 5 min at 14,000 rpm to purify the debris. Then 100 ml of 10% Triton X-100 and 30 ml of 5M NaCl were added. The sample was then incubated with 20 ml of Dynabeads Protein G (LifeTechnologies,10003D) for 1 h at 4°C. Primary antibodies were added to each tube and immunoprecipitation (IP) was conducted overnight in the cold room. Cross-linked complexes were precipitated with Dynabeads Protein G for 2 hr at 4°C. The beads were then washed in low salt wash buffer (20 mM Tris-HCl pH 8, 150 mM NaCl, 10 mM EDTA, and 1% SDS) for 5 min at 4°C, high salt wash buffer (50 mM Tris-HCl pH 8, 10 mM EDTA, and 1% SDS) for 5 min at 4°C and LiCl wash buffer (50 mM Tris-HCl pH 8, 10 mM EDTA, and 1% SDS) for 5 min at 4°C. DNA was eluted in elution buffer (100 mM sodium bicarbonate and 1% SDS). Cross-links were reversed overnight at 65°C. RNA and protein were digested with 0.2 mg/ml RNase A for 30 min at 37C followed by 0.2 mg/ml Proteinase K for 1 h at 55°C. DNA was purified with phenol-chloroform extraction and isopropanol precipitation.
ChIP-seq libraries were prepared using the Rubicon ThruPLEX DNA-seq Kit from 1 ng of purified ChIP DNA or input DNA according to the manufacturer’s protocol.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
genomic DNA immunoprecipitated without antibody
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| Data processing |
The adapter sequences were removed from the FASTQs using Trimmomatic (0.39).
The cleaned FASTQs were aligned using Bowtie2 (2.4.4) to the human reference genome GRCh38.
The duplicated reads were removed using picard (2.26.2) MarkDuplicates.
The bigWig files were generated using bamCoverage (3.5.1).
Assembly: GRCh38
Supplementary files format and content: bigWig
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| Submission date |
Jul 03, 2023 |
| Last update date |
Oct 31, 2023 |
| Contact name |
Masaki Nomura |
| E-mail(s) |
[email protected]
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| Organization name |
Foundation for Biomedical Research and Innovation at Kobe
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| Street address |
6-3-7 Minatojima Minamimachi, Chuo Ward
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| City |
Kobe |
| State/province |
Hyogo |
| ZIP/Postal code |
650-0047 |
| Country |
Japan |
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| Platform ID |
GPL24676 |
| Series (2) |
| GSE203322 |
BRD9 determines the cell fate of hematopoietic stem cells by regulating chromatin state |
| GSE236328 |
BRD9 determines the cell fate of hematopoietic stem cells by regulating chromatin state [human_Brd9_CTCF-ChIP-seq] |
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| Relations |
| BioSample |
SAMN36271118 |
| SRA |
SRX20866521 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7528023_Input_K562dBRD9_ready.bigWig |
44.0 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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