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GEO help: Mouse over screen elements for information. |
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Status |
Public on Sep 07, 2010 |
Title |
lung_RNAP-II_ChIP-Seq |
Sample type |
SRA |
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Source name |
Adult_Tissue
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Organism |
Mus musculus |
Characteristics |
tissue type: lung strain: FVB antibody: RNAP-II antibody manufacturer: Abcam antibody catalog number: ab5408
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Extracted molecule |
genomic DNA |
Extraction protocol |
About one gram of freshly dissected brain, kidney, liver, lung, or spleen tissue from FBV mice was minced finely and cross linked with 1% formaldehyde for 10 min at room temperature. To stop cross-linking glycine was added to a final concentration of 0.125 M. Next, the tissue sample was treated to isolate individual cells and cross-linked chromatin was fragmented to a size range of 0.2-0.8Kb. Chromatin immunoprecipitation was performed using 10µg of Pol-II antibody bound Dynal magnetic beads. The antibodies against Pol-II were purchased from Abcam Inc (ab5408). Following immunoprecipitation, the bound nucleoprotein complexes were extensively washed six times with wash buffer 1 and once with wash buffer 2 (Wash buffer 1: 50mM HEPES-KOH (pH7.55), 500mM LiCl, 1mM EDTA, 1.0% NP-40 and 0.7% Na-deoxycholate; Wash buffer 2: TE containing 50mM NaCl) and the ChIP enriched DNA was eluted and purified by phenol:chloroform:isoamyl alcohol extraction. This purified DNA (10ng) was further processed according to the Illumina Inc. instructions to prepare the library for sequencing ChIP enriched DNA.
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Library strategy |
ChIP-Seq |
Library source |
genomic |
Library selection |
ChIP |
Instrument model |
Illumina Genome Analyzer |
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Description |
Chromatin IP against RNAP-II
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Data processing |
Alignment: Alignment was performed using Bowtie and ELAND program
Peaks: Peak detection was performed using our three step procedure: (i) Identification of statistically significant sequence read enriched genomic regions (of length 1000bp). A region will be considered statistically significant if the number of reads within that region is higher than the number expected due to random background. We used Poisson distribution to estimate the background read count at a given significant level P (P <0.01). (ii) Creating the read overlapping profile for each identified region from step 1, by extending the sequence reads from the 5’ end to the 3’ end of the reads up to 400 bps (the average length of the ChIP-DNA fragment sequenced from the Solexa GA with Illumina standard ChIP-seq protocol). (iii) Peak identification – by counting the number of overlapped reads at each nucleotide position and defining the genomic position with the highest number as the peak position within the 1 kb significant region.
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Submission date |
May 11, 2010 |
Last update date |
May 15, 2019 |
Contact name |
Ravi Gupta |
E-mail(s) |
rgupta@wistar.org
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Phone |
215-495-6837
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Fax |
215-495-6847
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Organization name |
The Wistar Institute
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Department |
Center for Systems and Computational Biology, Molecular and Cellular Oncogenesis Program
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Lab |
Davuluri Lab
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Street address |
3601 Spruce Street
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City |
Philadelphia |
State/province |
PA |
ZIP/Postal code |
19104 |
Country |
USA |
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Platform ID |
GPL9185 |
Series (1) |
GSE21773 |
Genome-wide mapping of RNA Pol-II promoter usage in mouse tissues by ChIP-seq |
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Relations |
SRA |
SRX020252 |
BioSample |
SAMN00013377 |
Supplementary file |
Size |
Download |
File type/resource |
GSM542589_lung.bed.gz |
93.0 Mb |
(ftp)(http) |
BED |
GSM542589_lung.peak.txt.gz |
395.0 Kb |
(ftp)(http) |
TXT |
GSM542589_lung.wig.gz |
41.3 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
Processed data provided as supplementary file |
Raw data are available in SRA |
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