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| Status |
Public on Nov 15, 2016 |
| Title |
Cal-1 JQ1 IgG Control |
| Sample type |
SRA |
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| Source name |
Cal-1 JQ1 IgG Control
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| Organism |
Homo sapiens |
| Characteristics |
cell line: Cal-1
cell type: BPDCN (blastic plasmacytoid dendritic cell neoplasm) cells
disease state: BPDCN (blastic plasmacytoid dendritic cell neoplasm)
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| Treatment protocol |
Agent: JQ1
Treatment dose: 250 nM
Treatment time: 12 hours
In-vitro treatment: 1x10(8) exponentially growing cells were treated for 12 hours with 250 nM JQ1.
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| Growth protocol |
Cal-1 Growth Protocol
Other: Cal-1 cells were cultured in RPMI-1640 medium supplemented with penicillin/streptomycin and 10% fetal bovine serum (Tet tested, Atlanta Biologicals). All cell lines were maintained in a humidified, 5% CO2 incubator at 37C.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
IgG Control Extraction
Other: For "Super-enhancer" ChIP-Seq, 1x10(8) exponentially growing cells were collected by centrifugation, resuspended in room temperature RPMI without FBS ( 2x10(6) cells/ml) and cross-linked with 1% formaldehyde for 5 min at RT. Cross-linking was quenched by addition of 125 mM Glycine for 5 min at RT. Cross-linked cells were first washed with ice-cold PBS and then resuspended in ice-cold RIPA buffer (10mM Tris-HCl pH8, 140 mM NaCl, 1mM EDTA pH 8, 0.5 mM EGTA, 1% Triton X-100, 0.1% SDS and 0.1% Sodium Deoxycholate) to a final concentration of 5x10(6) cell/ml. DNA was sheared with a Misonix XL sonicator, by performing 12 x 45'' sonication cycles at power setting of 5. For the immune precipitation reaction, 2.0x10(7) chromatin cell equivalents were incubated overnight with 10 ug of Negative Control antibody (anti-Flag-M2, Sigma). The following day, chromatin/antibody complexes were incubated with 50 ul of Protein G magnetic beads (Invitrogen) for 4 h at 4C. Protein G bound complexes were washed 4 times with RIPA Buffer, once with LiCl Buffer (10 mM Tris-HCl pH8, 250 mM LiCl, 0.5% NP40, 0.5% Sodium Deoxycholate, 1 mM EDTA), once with TE pH 8.0 and finally resuspended in 100 ul TE pH8 containing RNAse A (0.2 ug/ul). Reverse cross-link was performed overnight at 65C, followed by treatment with 20 ug Proteinase K (Invitrogen) for 2 h at 50C. Final DNA purification was performed with QIAquick PCR Purification columns (QIAGEN).
ChIP DNA was used to generate ChIP-Seq libraries with the NEXTflexTM Illumina ChIP- Seq Library Prep Kit (Bio Scientific), according to manufacturer's instructions.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
No additional information.
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| Data processing |
BRD4 and RNA Pol2 ChIP-Seq Analyses
Calculation Method: For BRD4 and RNA Pol2 ChIP-Seq analyses, 36 bp sequence tags were aligned to human genome build 36 (hg18) with Bowtie software. Redundant reads were removed and reads uniquely mapping to reference genome were used for further analysis. A maximum of two mismatches was allowed for each read. For each gene, a representative RefSeq was defined based on the RNA Pol2 ChIP-Seq data. In the case of multiple RefSeq deriving from alternative promoter usage, the RefSeq with highest RNA Pol2 promoter density (-+ 500 bp from TSS) was selected. In the case of multiple RefSeq sharing the same promoter but differing for exon composition, the RefSeq with the overall highest RNA Pol2 density was selected. Promoter peaks were defined as peaks whose apex was located within a -+ 2Kb window from a representative TSS. Upstream binding peaks were defined as peaks whose apex was located within a -15Kb to -2Kb window from the TSS. Gene body binding peaks were defined as peaks whose apex was located in a window starting at +2Kb from the TSS and ending with the end of the RefSeq. When multiple peaks were associated with the same representative RefSeq, the peak with the highest apex was used for further analyses. To compare the results of two experiments, ChIP-Seq data were normalized by total tags count.
Supplementary_files_format_and_content: wig file
Genome_build: hg18
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| Submission date |
Dec 18, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Louis M. Staudt |
| E-mail(s) |
lstaudt@mail.nih.gov
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| Phone |
301-402-1892
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| Organization name |
National Cancer Institute
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| Department |
Lymphoid Malignancies Branch
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| Lab |
Louis M Staudt
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| Street address |
9000 Rockville Pike, Bldg 10, Rm 4N114
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| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
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| Platform ID |
GPL18573 |
| Series (1) |
| GSE76147 |
A druggable TCF4- and BRD4-dependent transcriptional network sustains malignancy in blastic plasmacytoid dendritic cell neoplasm (ChIP-Seq) |
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| Relations |
| BioSample |
SAMN04350635 |
| SRA |
SRX1491256 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1975024_Cal-1_JQ1_IgG_Control.wig.gz |
675.6 Kb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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