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Sample GSM249006 Query DataSets for GSM249006
Status Public on Dec 13, 2007
Title Undiluted spike-in rep2_B2_T1
Sample type genomic
 
Source name Human genomic DNA
Organism Homo sapiens
Characteristics Undiluted spike-in rep2
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 standard Affymetrix protocol
 
Hybridization protocol standard Affymetrix protocol
Scan protocol standard Affymetrix protocol
Description hybridized at Affymetrix
Data processing Model-based analysis of tiling array (MAT) chip.dfci.harvard.edu/~wli/MAT/
 
Submission date Dec 12, 2007
Last update date Aug 14, 2011
Contact name Wei Li
E-mail(s) WL1@bcm.edu
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)
GSE9848 Systematic evaluation of variability in simulated ChIP-chip experiments Kevin_Encode
GSE10114 Systematic evaluation of variability in ChIP-chip experiments using predefined DNA targets

Supplementary file Size Download File type/resource
GSM249006_ASCII.CEL.gz 31.4 Mb (ftp)(http) CEL
Processed data are available on Series record

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