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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jul 26, 2019 |
| Title |
Gata4.d2.FVF.Gata.r2 |
| Sample type |
SRA |
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| Source name |
ES cells
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| Organism |
Mus musculus |
| Characteristics |
cell line: doxycycline (Dox) inducible Foxa2-Venus, Gata4-tBFP ES cell line (T134)
genotype: TetON-Foxa2-Venus; TetON-Gata4-tBFP
facs sorting markers: Venus-positive/tBFP-positive
chip antibody: anti-Gata4 (R&D Systems; AF2606)
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
1-2 million FACS-sorted cross-linked cells (1% formaldehyde, 10min RT) were lysed in 100 ul Buffer-B-0.3 (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 0,3%SDS, 1x protease inhibitors -Roche) and sonicated in a microtube (Covaris; 520045) using a Covaris S220 device until most of the DNA fragments were 200-500 base pairs long (settings: temperature 4°C, duty cycle 2%, peak incident power 105 Watts, cycles per burst 200). After shearing, lysates were centrifuged 10min, 4°C, 12000g and supernatant diluted with 1 volume of Dilution Buffer (1mM EGTA 300 mM NaCl, 2% Triton x-100, 0.2% sodium deoxycholate, 1x protease inhibitors-Roche). Sonicated chromatin was then incubated 4h at 4°C on a rotating wheel with 6 ug of antibody conjugated to 20 µl of magnetic beads (Dynabeads, Life Technology). Beads were washed four times with Buffer-A (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.5 mM EGTA,1% Triton X-100, 0.1% SDS, 0.1% Na-deoxycholate, 140 mM NaCl, 1x protease inhibitors) and once with Buffer-C (10 mM Tris-HCl, pH 8.0, 10 mM EDTA). Beads were then incubated with 70 μl elution buffer (0.5% SDS, 300 mM NaCl, 5 mM EDTA, 10 mM Tris HCl pH 8.0) containing 2 μl of Proteinase K (20mg/ml) for 1 hour at 55°C and 8 hours at 65°C to revert formaldehyde crosslinking, and supernatant was transferred to a new tube. Another 30 μl of elution buffer was added to the beads for 1 minute and eluates were combined and incubated with another 1 μl of Proteinase K for 1 hour at 55°C. Finally, DNA was purified with SPRI AMPure XP beads (Beckman Coulter) (sample-to-beads ratio 1:2). Purified DNA was used as input for library preparation.
MicroPlex Library Preparation Kit v2 (Diagenode, cat. C05010012)
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 1500 |
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| Description |
ES cells were seeded without feedeers in gelatin-coated plates and grown for 48h in ES standard medium (LIF/serum) supplemented with 40ng/ml doxycycline.
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| Data processing |
reads were mapped to the mouse genome (mm10) using bowtie (Langmead et al., 2009)
Genome_build: mm10
Supplementary_files_format_and_content: bigWig files were generated from Homer tag directories using makeBigWig.pl
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| Submission date |
Feb 11, 2019 |
| Last update date |
Sep 02, 2019 |
| Contact name |
Filippo M. Cernilogar |
| E-mail(s) |
filippo.cernilogar@med.uni-muenchen.de
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| Organization name |
LMU Munich
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| Department |
Biomedical Center
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| Street address |
Grosshaderner Strasse 9
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| City |
Planegg-Martinsried |
| ZIP/Postal code |
82152 |
| Country |
Germany |
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| Platform ID |
GPL18480 |
| Series (2) |
| GSE116258 |
Pre-marked chromatin and transcription factor co-binding shape the pioneering activity of Foxa2 [ChIP-seq Transcription Factors] |
| GSE116262 |
Pre-marked chromatin and transcription factor co-binding shape the pioneering activity of Foxa2 |
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| Relations |
| BioSample |
SAMN10910715 |
| SRA |
SRX5360112 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3597748_Gata4.d2.FVF.Gata.r2_m1.ucsc.bigWig |
235.6 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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