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Sample GSM3223351 Query DataSets for GSM3223351
Status Public on Jul 26, 2019
Title MeDIP.5hmC.d3F
Sample type SRA
Source name mesendoderm cell
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-pos/Sox17-Cherry-neg
time point: d3
antibody: 5-Hydroxymethylcytosine (Active motif 39769)
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.
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).
Library strategy OTHER
Library source genomic
Library selection other
Instrument model Illumina HiSeq 1500
Data processing Library strategy: hMeDIP-seq
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
Submission date Jun 26, 2018
Last update date Aug 12, 2019
Contact name Filippo M. Cernilogar
Organization name LMU Munich
Department Biomedical Center
Street address Grosshaderner Strasse 9
City Planegg-Martinsried
ZIP/Postal code 82152
Country Germany
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
BioSample SAMN09486970
SRA SRX4298551

Supplementary file Size Download File type/resource
GSM3223351_MeDIP.5hmC.d3F.r1_m1.ucsc.bigWig 320.0 Mb (ftp)(http) BIGWIG
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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