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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Apr 01, 2013 |
| Title |
MmES_CMV_MeCP2 |
| Sample type |
SRA |
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| Source name |
embryonic stem cells
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| Organism |
Mus musculus |
| Characteristics |
cell type: embryonic stem cells
genotype: wild type
background strain: mixed 129-C57Bl/6
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| Growth protocol |
Wild type embryonic stem cells derived from mixed 129-C57Bl/6 background blastocysts, were cultivated on feeder cells or 0.2% gelatine coated dishes. ES cell growth medium consisted of DMEM (Invitrogen) supplemented with 15% foetal calf serum (Invitrogen), 1x non-essential amino acids (Invitrogen), 1mM L-glutamine, LIF and 0.001% beta-mercaptoethanol. Differentiation was performed as previously described (Bibel et al).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For cross-linking and chromatin extraction, cells were fixed for 10 minutes with 1% Formaldehyde at room temperature and incubated for 10 min on ice in presence of 1.2M Glycine. Cells were harvested and treated for 10 min with 10mM EDTA, 10mM TRIS, 0.5mM EGTA and 0.25% Triton X-100 and 10 min in 1mM EDTA, 10mM TRIS, 0.5mM EGTA and 200mM NaCl with subsequent lysis in 50mM HEPES, 1mM EDTA, 1% Triton X-100, 0.1% deoxycholate, 0.1% SDS and 150 mM NaCl for 2 hours on ice. Crosslinked chromatin was subjected to sonication in a Bioruptor instrument (Diagenode). ProteinA-dynabeads pre-cleared 150-250 µg chromatin were incubated with 40 µl blocked (1%CFSG, 100ng tRNA) Streptavidin-M280 magnetic beads over night at 4°C. Beads were washed with two rounds of 2% SDS, 1x high salt buffer, 1x LiCl Buffer and two rounds of TE. Beads were treated with RNaseA for 30 min at 37°C, Proteinase K for 3 hours at 50°C then de-crosslinked over night at 55°C. DNA was purified with Phenol-Chloroform extraction and EtOH precipitation. ChIP-seq with inline barcodes. Four libraries with different barcodes were pooled at equimolar ratios per lane.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
basecalls using CASAVA
fastq to fasta conversion
reads from pooled libraries were demultiplexed using Barcodesplitter from FASTX tools, barcodes were removed
reads were aligned to mm9 mouse genome using BOWTIE, allowing up to 2 missmatches
coordinates of reads that map only once to the mouse genome were converted to BED format
Genome_build: mm9
Supplementary_files_format_and_content: bed format, contents: chromosome, start, end, read_name, n times mapped to genome, strand
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| Submission date |
Feb 04, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Dirk Schuebeler |
| Organization name |
Friedrich Miescher Institute for Biomedical Research
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| Street address |
Maulbeerstrasse 66
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| City |
Basel |
| ZIP/Postal code |
4058 |
| Country |
Switzerland |
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| Platform ID |
GPL13112 |
| Series (1) |
| GSE39610 |
Methylation-dependent and -independent genomic targeting principles of the MBD protein family |
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| Relations |
| SRA |
SRX224514 |
| BioSample |
SAMN01909374 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1077270_MmES_CMV_MeCP2.bed.gz |
307.7 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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