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Sample GSM249014 Query DataSets for GSM249014
Status Public on Dec 13, 2007
Title Spike-in rep1
Sample type genomic
Source name Human genomic DNA
Organism Homo sapiens
Characteristics Spike-in
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.
Label biotin
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
Hybridization protocol standard Affymetrix protocol
Scan protocol standard Affymetrix protocol
Description Spike-in rep1
Data processing Model-based analysis of tiling array (MAT)
Submission date Dec 12, 2007
Last update date Aug 14, 2011
Contact name Wei Li
Organization name Baylor College of Medicine
Street address one baylor plaza, MS BCM305
City Houston
State/province TX
ZIP/Postal code 77030
Country USA
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

Supplementary file Size Download File type/resource
GSM249014.CEL.gz 7.3 Mb (ftp)(http) CEL
GSM249014_ASCII.CEL.gz 11.3 Mb (ftp)(http) CEL
Processed data are available on Series record

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