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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Feb 01, 2013 |
| Title |
9A_embryo-dko |
| Sample type |
SRA |
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| Source name |
brain
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| Organism |
Mus musculus |
| Characteristics |
tissue: brain
developmental stage: embryo
strain: mixed 129|C57BL/6
tet1/tet2 treatment: TET1 and TET2 knocked out
antibody: 5-methylcytosine
vendor/catalog/lot: monoclonal, Active Motif, 39649
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Homogenized mouse tissues were incubated overnight in 100mM Tris-HCl pH 8 containing 5mM EDTA, 0.2% SDS, 200 mM NaCl and 400 µg/ml proteinase K, and DNA was then isolated using a standard protocol with phenol:chloroform:isoamyl alcohol and Phase Lock Gel™ separation tubes. Genomic DNA was finally treated with RNAse A (20 µg/ml) for 30 minutes at 37°C.
Thirty µg of genomic DNA for each sample was sonicated to 300bp fragments using a Covaris S220 (Covaris). Sheared DNA was ethanol precipitated, washed with 70% ethanol, and dissolved in TE buffer (pH8.0). Illumina adapters were ligated using the End-It DNA End-repair kit (Epicentre). DNA was purified with the QiaQuick PCR purification kit (Qiagen) and diluted to 122.5µl with ddH2O. After the addition of 15µl NEB buffer 2, 3.5µl of 10mM ATP and 9µl of 5U/µl Klenow exo- (NEB), reactions were incubated at 37°C for 50min. DNA was subsequently purified with the QiaQuick PCR purification kit, diluted to 24µl with ddH2O, and mixed with 50µl of 2x NEB quick ligation buffer, 20 µl of barcoded Illumina adapter and 6µl of quick T4 DNA ligase (NEB). After 20 minutes incubation at room temperature, DNA was purified again with QiaQuick PCR purification kit. Ligation efficiency was analyzed by PCR on 10ng of final ligated DNA with Illumina sequencing primers. Finally, five µg of adapter ligated DNA was used for the immunoprecipitation, as described above. Immunoprecipitated DNA was resolved in 20µl of water and all (h)MeDIP samples were pooled to create the MeDIP and hMeDIP libraries, respectively. Both libraries were purified on e-Gel (Invitrogen) and the 300bp bands (containing roughly 200bp fragments of adapter-ligated immunopurified DNA) were extracted. Libraries were amplified using Illumina-Enrichment Primers for 12 cycles and purified using Agencourt AMPure XP beads (Beckman Coulter). DNA concentrations were measured using a Agilent High Sensitivity DNA Assay (Agilent Technologies) and 7 pmol were used for cluster PCR and substantial 100bp paired-end sequencing on a HiSeq2000 (Illumina).
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| Library strategy |
MeDIP-Seq |
| Library source |
genomic |
| Library selection |
5-methylcytidine antibody |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
Strategy: MeDIP, Barcode: CGAAAC
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| Data processing |
Illumina Casava1.8.1 software used for basecalling.
Preprocessing, read trimming: shore 0.6.2
Preprocessing, quality filtering: shore 0.6.2
Mapping: bowtie
computation of coverage values: wiggles
Genome_build: mm9
Supplementary_files_format_and_content: Format: bedgraph, Content: genomic coverage
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| Submission date |
Nov 19, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Guenter Raddatz |
| Organization name |
German Center for Cancer Research
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| Street address |
Im Neuenheimer Feld 580
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| City |
Heidelberg |
| ZIP/Postal code |
69120 |
| Country |
Germany |
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| Platform ID |
GPL13112 |
| Series (1) |
| GSE42396 |
Combined deficiency of Tet1 and Tet2 is compatible with development but leads to epigenetic instability |
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| Relations |
| SRA |
SRX205303 |
| BioSample |
SAMN01816450 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1039183_9a.bedgraph.gz |
2.2 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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