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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Feb 28, 2017 |
| Title |
PDGF K27M EPZ me3 rep2 |
| Sample type |
SRA |
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| Source name |
PDGF Neural Stem Cells
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| Organism |
Mus musculus |
| Characteristics |
cell type: PDGF Neural Stem Cells
genotype: K27M
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| Treatment protocol |
The cells were treated with either DMSO or Ezh2 inhibitor (EPZ6438, 3 uM) for 12 days.
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| Growth protocol |
Mouse cells were cultured on poly-D-lysine (PDL, Sigma) and laminin (Sigma) coated plates in neural stem cell medium (50% DMEM-F12, 50% neurobasal medium, N2 and B27 supplements, sodium pyruvate, glutamax, HEPES, β−mercaptoethanol, non-essential amino acids, bovine serum albumin, heparin, 100U/ml penicillin, 100 μg/ml streptomycin, human recombinant epidermal and basic fibroblast growth factors).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were cross-linked with 1% formaldehyde for 10 minutes at room temperature. Glycine was added at a final concentration of 125 mM to quench the formaldehyde. Cells were then washed twice with PBS and harvested in SDS buffer (50 mM Tris at pH 8.1, 0.5% SDS, 100 mM NaCl, 5 mM EDTA). Cells were pelleted down, resuspended in Triton-X IP buffer (100 mM Tris at pH 8.6, 0.3% SDS, 1.7% Triton X-100, and 5 mM EDTA) and the chromatin was sonicated to get the DNA fragments of <1000 bp with average DNA fragment size of 300 bp.
500 μg chromatin was used to perform chromatin immunoprecipitation (ChIP) and 10 ng of ChIP DNA was used to make library using NEBNext Ultra DNA library kit for illumine (E7370; New England Biolabs) according to supplier’s instructions. The libraries were sequenced using Illumina HiSeq2500 sequencer.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
treatment: EPZ
ChIP: H3K27me3 (Cell Signaling; catalog # C36B11)
replicate: R2
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| Data processing |
Reads were mapped to the mouse (UCSC mm10) genome assembly using bowtie (version 1.1.2) with default parameters except concerning multi-mapping reads which were discarded using option "-m1". For each library, mapped reads were de-duplicated by keeping a single read in each group of reads mapping to the exact same genomic sequence.
To generate ChIP-seq profiles, reads were extended to a total length of 180 bp starting from the 5' end, representing the average DNA fragment length in our libraries, and genomic coverage was computed at base pair resolution. For each library, coverage values were scaled according to the estimated Background Read Density (BRD, see the methods description in our study).
Regions locally enriched for H3K27me3, Suz12 and Ring1b were established with the SPP peak calling package using the broad enrichment procedure and IgG samples as control. We used default parameters except a z-threshold value of 6.85 and background weights corresponding to our estimated BRD scaling for the normalization between ChIP and IgG libraries.
Genome_build: mm10
Supplementary_files_format_and_content: BED files with SPP peaks, bigWig files with normalized ChIP-seq profiles
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| Submission date |
Aug 09, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Faizaan Mohammad |
| E-mail(s) |
faizaan.mohammad@bric.ku.dk
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| Organization name |
BRIC
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| Street address |
Ole Maaloøs vej 5 Fourth Floor
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| City |
Copenhagen |
| State/province |
Choose A State |
| ZIP/Postal code |
2200 |
| Country |
Denmark |
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| Platform ID |
GPL17021 |
| Series (2) |
| GSE85386 |
EZH2 is a potential therapeutic target for H3K27M mutant paediatric gliomas [ChIP-Seq mouse cell lines] |
| GSE85390 |
EZH2 is a potential therapeutic target for H3K27M mutant paediatric gliomas |
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| Relations |
| BioSample |
SAMN05552976 |
| SRA |
SRX2010474 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2266123_NSC_PDGF_K27M_EPZ_H3K27me3_R2.bed.gz |
3.2 Kb |
(ftp)(http) |
BED |
| GSM2266123_NSC_PDGF_K27M_EPZ_H3K27me3_R2.bw |
177.6 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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