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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Mar 26, 2012 |
| Title |
MM control PvuII |
| Sample type |
SRA |
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| Source name |
MM_control_PvuII_seq
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| Organism |
Mus musculus |
| Characteristics |
cell type: mouse embryonic fibroblasts
genotype/variation: untransduced control
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| Treatment protocol |
P19 cells and MEFs were infected with lentivirus in media containing polybrene (final concentration 8 µg /ml), after 24 hours the cells were switched into fresh media. MEFs transduced with MyoD were switched to differentiation media (1% heat inactivated horse serum, 10 μg/mL insulin, 10 μg/mL transferrin) 48 hours after infection.
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| Growth protocol |
P19 cells were maintained in MEMα as previously described (Farah et al., 2000, PUBMED ID 10648228), and MEFs derived from Myod-/-/Myf5-/- mice were maintained in DMEM with 10% FBS.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Standard Illumina ChIP-Seq sample prep kit protocol with two modifications: (1) DNA fragments ranging from 150 to 300 bp were selected at the gel-selection step; (2) 21 cycles of PCR were performed at the PCR amplification step instead of 18.
For P19 and MM control PvuII seq sample, untranduced P19 or MEFs cells were trypsinized and washed once in reticulocyte suspension buffer (RSB: 10mM Tris pH 7.4, 10mM NaCl, 5mM MgCl2), followed by resuspension in lysis buffer (RSB + 0.1% NP-40) at a final concentration of 1.5x106 cells/mL and incubation on ice for 10 minutes. Nuclei were pelleted and washed in lysis buffer, followed by resuspension in 200μl of 1X NEB buffer 2, addition of 40 units of PvuII (NEB) per 106 nuclei, and incubation at 37C for 30 minutes. 200μl of STOP buffer (0.6M NaCl, 20mM Tris pH 7.4,10mM EDTA, 1% SDS, 2 mg/mL proteinase K) was added and the reaction was incubated at 37C overnight. Genomic DNA was isolated using Qiagen DNeasy spin columns. 5μg of DNA was used for labeling, beginning with addition of an 'A' tail to the blunt ends generated by PvuII digestion using Klenow 3-5' exo- (NEB). After purification through MinElute columns (Qiagen), custom designed biotinylated adapters with a 'T' overhang and an EcoRV site immediately upstream of the 'T' (purchased from IDT) were ligated onto the 'A'-tailed ends using Quick Ligase (NEB). After purification (Qiagen), DNA was fragmented to 150-350bp using a Diagenode Bioruptor (low amplitude, 30 seconds/cycle, 30 cycles). Biotinylated fragments were enriched with Streptavidin-conjugated Dynabeads (Invitrogen), and DNA was released from the beads by digestion with EcoRV for 1 hour at 37C. Fragments were subsequently purified and labeled for sequencing as above.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Description |
chromatin accessibility using PvuII assay followed by high throughput sequencing
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| Data processing |
MM.control.P2.bam; genome build: mm9
Peaks are called using in-house developed R package peakSig.
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| Submission date |
Jan 06, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Stephen Tapscott |
| E-mail(s) |
stapscot@fredhutch.org
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| Organization name |
Fred Hutch Cancer Research Center
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| Department |
Human Biology
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| Lab |
Tapscott
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| Street address |
1100 Fairview N. Ave
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| City |
Seattle |
| State/province |
WASHINGTON |
| ZIP/Postal code |
98103 |
| Country |
USA |
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| Platform ID |
GPL11002 |
| Series (2) |
| GSE34906 |
Genetic and epigenetic determinants of neurogenesis and myogenesis [ChIP-seq] |
| GSE34908 |
Genetic and epigenetic determinants of neurogenesis and myogenesis |
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| Relations |
| SRA |
SRX114687 |
| BioSample |
SAMN00770009 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM857394_MM.control.P2.bam |
669.9 Mb |
(ftp)(http) |
BAM |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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