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| Status |
Public on Jul 26, 2019 |
| Title |
Nanog.d0.r1 |
| Sample type |
SRA |
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| Source name |
ES cells
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| Organism |
Mus musculus |
| Characteristics |
cell line: double knock-in (DKI) mouse ES cell line carrying Foxa2-Venus and Sox17-Cherry gene fusions
genotype/variation: Foxa2-Venus heterozygous;Sox17-Cherry homozygous
facs sorting markers: Foxa2-Venus-neg/Sox17-Cherry-neg
time point: d0
chip antibody: anti-Nanog (Bethyl lab; A300-397-A)
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| Growth protocol |
The cells were cultured for few consecutive passages on gelatine and ES medium. On the day of differentiation, mESCs were seeded on 10 cm gelatine coated dishes directly in endoderm differentiation medium (EDM) supplemented with 1 ng/ml of murine Wnt3a and 10 ng/ml of Activin A. Freshly prepared EDM supplemented with Wnt3a and Activin A was added every day. Cells were collected on day0, day3 and day5 for FACS isolation.
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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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| Data processing |
Reads were mapped to the mouse genome (mm10) using bowtie (Langmead et al., 2009).
Genome_build: mm10 (GRCm38)
Supplementary_files_format_and_content: bigWig files were generated from Homer tag directories using makeBigWig.pl.
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| Submission date |
Jun 25, 2018 |
| Last update date |
Aug 12, 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 |
SAMN09486918 |
| SRA |
SRX4298475 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3223331_Nanog.d0.r1_m1.ucsc.bigWig |
250.0 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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