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| Status |
Public on Dec 13, 2007 |
| Title |
Spike-in rep3 |
| Sample type |
genomic |
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| Source name |
Human genomic DNA
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| Organism |
Homo sapiens |
| Characteristics |
Spike-in
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
To create our simulated ChIP spike-in mixture, we first randomly selected 100 cloned genomic DNA sequences (average length 497 bp) corresponding to predicted promoters in the human genome15, individually purified them, and normalized the concentrations of each preparation to 500 pg/µL (Figure 1). To create enrichment levels that ranged from 1.25- to 196-fold relative to genomic DNA (Supplemental Tables 1 and 2), we added the appropriate volume of these stock solutions to a commercial human genomic DNA preparation (Methods; Supplemental Tables 1 and 2). The clones were validated by sequencing and PCR both before and after dilution (Supplemental Methods). We prepared one clone mixture to be directly labeled and hybridized to arrays at the given concentration ("undiluted", 77 ng/µL), and a different clone mixture that was diluted such that amplification would be necessary before labeling and hybridization ("diluted", 3 ng/µL). The diluted mixture was created because all of the array platforms require microgram quantities of DNA and a typical ChIP experiment produces ~50 ng of DNA, making amplification essential for most ChIP-chip experiments. Each amplification method is known to cause under- and over-representation of certain sequences16, which we aimed to assess in this context.
After the mixtures were prepared, the clones and their relative concentrations were again validated by PCR and quantitative PCR (qPCR). Note that the while the same spike-in clones were present in the diluted and undiluted mixtures, they were used at different enrichment levels in the two samples. In each mixture, most of the selected enrichment levels were represented by 10 distinct clones. To challenge the sensitivity of the array technologies, spike-in enrichment levels were biased towards enrichment levels less than tenfold. We also prepared two samples containing genomic DNA at 77 ng/µL and 3 ng/µL respectively without any spike-ins to serve as controls. We sheared the DNA mixtures with a standard chromatin sonication procedure17.
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| Label |
biotin
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| Label protocol |
On ice: - Dilute DnaseI (Ambion) to appropriate amount based on optimized conditions. We do a 1:100 dilution in water and use 1ul of this for each sample. Include the buffer. Efficient digestion can be checked by running the DNA on a gel. The optimal smear should range from 50bp to 100bp. A little higher is OK. - So, add buffer, Dnase I and buffer to final volume of 55ul. Run the program: 37C 30 min 95C 15 min 4C hold - Run 5ul on a gel to check for good fragmentation. Smear should be between 50bp and 100bp, although a little larger is OK On ice: - Add: 13ul TdT buffer (Promega) 1ul Biotin (1mM stock) 1ul TdT (30U/ul) 50ul of fragmented DNA -Run the program: 37C 16 hours 95C 10 min 4C hold - Spin briefly and prepare for hybridization
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| Hybridization protocol |
standard Affymetrix protocol
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| Scan protocol |
standard Affymetrix protocol
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| Description |
Spike-in rep3
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| Data processing |
Model-based analysis of tiling array (MAT) chip.dfci.harvard.edu/~wli/MAT/
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| Submission date |
Dec 12, 2007 |
| Last update date |
Aug 14, 2011 |
| Contact name |
Wei Li |
| E-mail(s) |
WL1@bcm.edu
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| Organization name |
Baylor College of Medicine
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| Street address |
one baylor plaza, MS BCM305
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| City |
Houston |
| State/province |
TX |
| ZIP/Postal code |
77030 |
| Country |
USA |
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| Platform ID |
GPL6129 |
| Series (2) |
| GSE9849 |
Systematic evaluation of variability in simulated ChIP-chip experiments Myles_Encode |
| GSE10114 |
Systematic evaluation of variability in ChIP-chip experiments using predefined DNA targets |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM249016.CEL.gz |
7.2 Mb |
(ftp)(http) |
CEL |
| GSM249016_ASCII.CEL.gz |
11.2 Mb |
(ftp)(http) |
CEL |
| Processed data are available on Series record |
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