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| Status |
Public on Jul 26, 2019 |
| Title |
MeDIP.5mC.d0 |
| 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
antibody: anti-5-methylcytosine (Eurogentec BI-MECY-0100)
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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 |
Genomic DNA from FACS-sorted cells was randomly sheared to 100-500 bp in a microtube (Covaris; 520045) using a Covaris S220 device (400 sec; 4°C; PP 140; DF 10; CB 200). Sonicated DNA was end-repaired, A-tailed, and ligated to Illumina multiplex adaptors according to NEBNext DNA library prep kit (NEB E6040S). Ligated DNA was purified using Agencourt AMPure XP beads (Beckman Coulter). 1 µg of adaptor-ligated DNA was used for each immunoprecipitation and heat-denatured at 95°C for 10min, rapidly cooled on ice and immunoprecipitated overnight at 4 ˚C with rocking agitation in 500 ml immunoprecipitation buffer (10mM sodium phosphate buffer, pH 7.0, 140mM NaCl, 0.05% Triton X-100) using 1 µl of mouse monoclonal anti-5-methylcytosine antibody (Eurogentec BI-MECY-0100) or 0.5 µl of rabbit 5-Hydroxymethylcytosine antibody (Active motif 39769). To recover the antibody-bound DNA fragments, 50µl Protein G Dynabeads (ThermoFisher) and, only to anti-5-methylcytosine IPs, 5µl of rabbit anti-mouse IgG secondary antibody (Active Motif cat.53017) were added and incubated for an additional 2 h at 4 ˚C with agitation. After immunoprecipitation, a total of seven to ten immunoprecipitation washes were performed with ice-cold immunoprecipitation buffer. Washed beads were resuspended in TE buffer with 0.25% SDS and 0.25 mg/ml proteinase K for 2 h at 55 ˚C with vigorous shaking (900 rpm). DNA was purified with the Qiagen PCR clean-up MinElute kit (Qiagen) and eluted in 30 ul. Samples were then amplified by PCR with Illumina primers (NEBNext Multiplex Oligos for Illumina cat. E7335) in a 50 μl reaction with 2× PCR master mix (NEB cat. M0541). PCR cycled as: (1) 98 °C, 30s; (2) 98 °C, 10 s; (3) 60 °C, 30 s; (4) 72 °C, 30s; (5) repeat steps (2)–(4) for 4-10 cycles; (6) 72°C, 5min. Amplified libraries were purified using Agencourt AMPure XP beads (Beckman Coulter). Quality control was carried out with a Qubit fluorometer and a Bioanalyzer (Agilent).
NEBNext DNA library prep kit (NEB E6040S).
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| Library strategy |
MeDIP-Seq |
| Library source |
genomic |
| Library selection |
5-methylcytidine antibody |
| 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 26, 2018 |
| Last update date |
Aug 12, 2019 |
| Contact name |
Filippo M. Cernilogar |
| E-mail(s) |
[email protected]
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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) |
| GSE116261 |
Pre-marked chromatin and transcription factor co-binding shape the pioneering activity of Foxa2 [MeDIP-seq] |
| GSE116262 |
Pre-marked chromatin and transcription factor co-binding shape the pioneering activity of Foxa2 |
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| Relations |
| BioSample |
SAMN09486968 |
| SRA |
SRX4298553 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3223353_MeDIP.5mC.d0.r1_m1.ucsc.bigWig |
361.9 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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