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| Status |
Public on Oct 21, 2016 |
| Title |
TEAD4 d1BC minus dox |
| Sample type |
SRA |
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| Source name |
TEAD4 d1BC minus dox
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| Organism |
Mus musculus |
| Characteristics |
cell type: d1 blast culture Flk1+ IVD cells
dox treatment: 0d
chip antibody: TEAD4 (Abcam, catalog# ab58310)
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| Treatment protocol |
cells were treated with 1µg/ml doxycycline for the duration indicated in each experiment.
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| Growth protocol |
A Flag-tagged dominant negative FOS (dnFOS) construct was PCR-amplified from CMV plasmid (CMV500-8584hep-fosLZ(MO), A-FOS, kindly provided by Charles Vinson (Olive et al, 1997)) and HindIII as well as Not1 sites were introduced. The GFP of the p2lox plasmid (kindly provided by Michael Kyba (Kyba et al, 2002)) was exchanged for Flag-tagged dnFOS. A2lox ESCs (a gift from Michael Kyba (Kyba et al, 2002)) or Bry-GFP ESCs were cultured as described before (Gilmour et al, 2014; Regha et al, 2015). Briefly, cells were grown on mitomycin C-inactivated mouse embryonic feeder cells (MEFs) in DMEM medium supplemented with 15% FCS (Stem Cell Technologies), 1 mM sodium pyruvate, 1 mM glutamine, 100 units per ml penicillin and 100 μg ml−1 streptomycin, 25 mM HEPES buffer, 1 × non-essential amino acids, 0.15 mM MTG and 103 units per ml LIF (ESGRO mLIF, Millipore ESG1107). Medium was changed every day and cells were passaged every 2nd day using TrypLE Express (Thermo Fisher). For transfection with p2lox-dnFOS plasmid, 2.5x106 A2lox ESCs were resuspended in 100 µl PBS (Sigma D8537), 20 µg of each p2lox-dnFOS plasmid and Cre-expressing plasmid were added and cells were electroporated at 240 V, 7 ms settings on a EPI 2500 electroporator (Fischer). Cells were plated quickly on NEO-resistant feeder cells in ESC medium and 1 day later selection was started by adding 300µg/ml G418. Clones were picked after 6 days of selection, assessed for their differentiation ability and transgene expression upon doxycycline addition. ESCs were cultured for one day on gelatinized plates in DMEM ESC medium and for another day on gelatinized plates in IMDM ESC medium in the presence of LIF. Subsequently, cells were differentiated as embryoid bodies (EBs) in 15cm bacterial-grade dishes at 1.25x106 cells per 50 ml for 3.75 days in IMDM differentiation medium without LIF. EBs were then dispersed to single cells by TrypLE Express treatment and Flk1pos cells were purified using MACS technology. Purified Flk1pos cells were cultured in gelatinized T150 tissue culture-grade flask at 1.2x106 cells per flask in blast culture medium (IMDM supplemented with 10% FCS, 100 units per ml penicillin and 100 μg ml−1 streptomycin, 1 mM glutamine, 0.45 mM MTG, 0.18 mg ml−1 Human transferrin (Roche 10652202001) 25 μg ml−1 ascorbic acid, 20% D4T conditioned media, 5 μg l−1 recombinant mouse Vascular Endothelial Growth Factor (Peprotech: 450-32), 10 μg l−1 mIL-6 (Peprotech: 216–16)).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin was prepared and immunoprecipitation was carried out as previously described (Lichtinger et al). Cells were resuspended in PBS at 3.3x106 cells/ml and cross-linked with 0.83mg/ml disuccinimidyl glutarate (DSG) for 45 minutes at room temperature with rotation. Cells were washed four times with PBS and further crosslinked in PBS at 2x106 cells/ml with 1% formaldehyde for 10 minutes at room temperature. Crosslinking was quenched with 0.125M glycine, cells were washed twice in PBS and incubated with Buffer A at 4oC for 10 minutes followed by buffer B at 4oC for 10 minutes. Double cross-linked chromatin was then used for immunoprecipitation as previously described (Lichtinger et al). Antibodies used were for the antibodies, Fos (sc-253-X), Jun (sc-1694-X) and TEAD4 (ab58310).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Base-calling (CASAVA)
Demultiplexing (bcl2fastq)
ChIP-seq reads were aligned to the mm10 genome assembly using Bowtie 1.00 to sorted BAM via stdoutput using --all --best --strata --v 2 -m 1 -S | samtools view -S - -b | samtools sort -
Tag shift (macs14)
Wig coverage track generation (macs14)
Peak detection (macs14)
Genome_build: mm10
Supplementary_files_format_and_content: Variable step 10bp wig files depicting ChIP-Seq coverage
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| Submission date |
Mar 16, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Pierre Daniel Cauchy |
| E-mail(s) |
cauchy@ie-freiburg.mpg.de
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| Phone |
+49 (0)761 270-77576
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| Organization name |
University Medical Center Freiburg
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| Department |
Zentrum für Translationale Zellforschung
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| Lab |
AG Onco-Immunology
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| Street address |
Breisacher Str. 115
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| City |
Freiburg |
| State/province |
Baden-Württemberg |
| ZIP/Postal code |
79106 |
| Country |
Germany |
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| Platform ID |
GPL17021 |
| Series (2) |
| GSE79320 |
AP-1 and TEAD4 co-operatively promote the vascular program during hemangioblast specification [ChIP-Seq] |
| GSE79323 |
AP-1 and TEAD4 co-operatively promote the vascular program during hemangioblast specification |
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| Relations |
| BioSample |
SAMN04562495 |
| SRA |
SRX1639172 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2091494_TEAD4_d1_BC_DNFOS_untreated_mm10_treat_afterfiting_all.wig.gz |
489.5 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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