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| Status |
Public on Nov 15, 2016 |
| Title |
Cal-1 TCF4 mAb |
| Sample type |
SRA |
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| Source name |
Cal-1 TCF4 mAb
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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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| 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 |
TCF4 mAb Extraction
Other: For the TCF4 ChIP-Seq, 5x10(7) 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 rabbit monoclonal TCF4 antibody developed by Epitomics. 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 Genome Analyzer IIx |
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| Description |
No additional information.
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| Data processing |
TCF4 ChIP-Seq Analysis
Calculation Method: For TCF4 ChIP-Seq analysis, 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. To perform "peak calling", the genome was divided into "bins" of 25 bp. Each sequencing tag was associated with the bin of its start site and an additional extension of 7 bins (for a total of 200bp) along the direction of its read to match the average library size. For a given experiment, the number of "hits" in a bin was defined as the number of extended tags associated with that bin. Bins with fewer than 3 hits were removed. Peaks were defined by merging consecutive significant bins. The height of the peak was defined as the largest number of hits in any of the bins forming the peak. The "apex" of the peak was defined as the bin that had this largest number of hits. In the case of ties, the earliest such peak was chosen as the apex. A threshold of apex >30 tags/bin was used to define high-stringency TCF4 binding peaks. A Negative Control IP (anti-Flag antibody) experiment was included as specificity control for each cell line. Experimental peaks overlapping with a control bin with a tag count of 5 or greater were excluded from further analysis. To compare the results of two experiments, ChIP-Seq data were normalized by total tags count.
Supplementary_files_format_and_content: bed 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 |
GPL10999 |
| 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 |
SAMN04350629 |
| SRA |
SRX1491250 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1975018_Cal-1_TCF4_mAb.bed.gz |
5.9 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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