Description

Each of these four tracks shows the binding sites for STAT1 ChIP-chip using Human Hela S3 cells hybridized to four different array designs/platforms. The first three platforms are custom maskless photolithographic arrays with oligonucleotides tiling most of the non-repetitive DNA sequence of the ENCODE regions:

• Maskless design #1: 50mer oligonucleotides tiled every 38 bps (overlapping by 12 nts)
• Maskless design #2: 36mer oligonucleotides tiled end to end
• Maskless design #3: 50mer oligonucleotides tiled end to end

The fourth array platform is an ENCODE PCR Amplicon array manufactured by Bing Ren's lab at UCSD.

Each track shows the combined results of multiple biological replicates: five for the first maskless array (50-mer every 38 bp), two for the second maskless array (36-mer every 36 bp), three for the third maskless array (50-mer every 50 bp) and six for the PCR Amplicon array. For all arrays, the STAT1 ChIP DNA was labeled with Cy5 and the control DNA was labeled with Cy3. See NCBI GEO GSE2714 for details of the experimental protocols.

Methods

Maskless photolithographic arrays

The data from replicates were median-scaled and quantile-normalized to each other (both Cy3 and Cy5 channels). Using a 501 bp sliding window centered on each oligonucleotide probe, a signal map (estimating the fold enrichment [log₂ scale] of ChIP DNA) was generated by computing the pseudomedian signal of all log₂(Cy5/Cy3) ratios (median of pairwise averages) within the window, including replicates. Using the same procedure, a -log₁₀(P-value) map (measuring significance of enrichment of oligonucleotide probes in the window) for all sliding windows was made by computing P-values using the Wilcoxon paired signed rank test comparing fluorensent intensity between Cy5 and Cy3 for each oligonucleotide probe (Cy5 and Cy3 signals from the same array). A binding site was determined by thresholding both on fold enrichment and -log₁₀(P-value) and requiring a maximum gap and a minimum run between oligonucleotide positions.

For the first maskless array (50-mer every 38 bp):
   log₂(Cy5/Cy3) >= 1.25, -log₁₀(P-value) >= 8.0, MaxGap <= 100 bp, MinRun >= 180 bp

For the second maskless array (36-mer every 36 bp):
   log₂(Cy5/Cy3) >= 0.25, -log₁₀(P-value) >= 4.0, MaxGap <= 250 bp, MinRun >= 0 bp

For the third maskless array (50-mer every 50 bp):
   log₂(Cy5/Cy3) >= 0.25, -log₁₀(P-value) >= 4.0, MaxGap <= 250 bp, MinRun >= 0 bp

PCR Amplicon Arrays

The Cy5 and Cy3 array data were loess-normalized between channels on the same slide and then between slides. A z-score was then determined for each PCR amplicon from the distribution of log(Cy5/Cy3) in a local log(Cy5*Cy3) intensity window (see Quackenbush, 2002 and the Express Yourself website for more details). From the z-score, a P-value was then associated with each PCR amplicon. Hits were determined using a 3 sigma threshold and requiring a spot to be present on three out of six arrays.

Verification

ChIP-chip binding sites were verified by comparing "hit lists" generated from combinations of different biological replicates. Only experiments that yielded a significant overlap (greater than 50 percent) were accepted. As an independent check (for maskless arrays), data on the microarray were randomized with respect to position and re-scored; significantly fewer hits (consistent with random noise) were generated this way.

Credits

This data was generated and analyzed by the labs of Michael Snyder, Mark Gerstein and Sherman Weissman at Yale University. The PCR Amplicon arrays were manufactured by Bing Ren's lab at UCSD.

References

Cawley, S., Bekiranov, S., Ng, H.H., Kapranov, P., Sekinger, E.A., Kampa, D., Piccolboni, A., Sementchenko, V., Cheng, J. et al. Unbiased mapping of transcription factor binding sites along human chromosomes 21 and 22 points to widespread regulation of noncoding RNAs. Cell 116(4), 499-509 (2004).

Euskirchen, G., Royce, T.E., Bertone, P., Martone, R., Rinn, J.L., Nelson, F.K., Sayward, F., Luscombe, N.M., Miller, P. et al. CREB binds to multiple loci on human chromosome 22, Mol Cell Biol. 24(9), 3804-14 (2004).

Luscombe, N.M., Royce, T.E., Bertone, P., Echols, N., Horak, C.E., Chang, J.T., Snyder, M. and Gerstein, M. ExpressYourself: A modular platform for processing and visualizing microarray data. Nucleic Acids Res. 31(13), 3477-82 (2003).

Martone, R., Euskirchen, G., Bertone, P., Hartman, S., Royce, T.E., Luscombe, N.M., Rinn, J.L., Nelson, F.K., Miller, P. et al. Distribution of NF-kappaB-binding sites across human chromosome 22. Proc Natl Acad Sci U S A. 100(21), 12247-52 (2003).

Quackenbush, J.. Microarray data normalization and transformation, Nat Genet. 32(Suppl), 496-501 (2002).