## Description

This track shows the map of -log10(P-value) for ChIP-chip using 3 antibodies in HeLa S3 (cervix epithelial adenocarcinoma) and GM06990 (lymphoblastoid) cells hybridized to maskless photolithographic arrays with 50-mer oligonucleotides tiled with 12-nt overlaps covering most of the non-repetitive DNA sequence of the ENCODE regions The antibodies used were:

- anti-RNA polymerase II ( N-20 polyclonal antibody)
- anti-RNA polymerase II ( 8WG16 monoclonal antibody against pre-initiation complex form)
- anti-H4Kac4 (Histone H4 tetra-acetylated lysine)

The initiation-complex form of RNA polymerase II is associated with the transcription start site and tetra-acetylated Histone H4 is found in transcriptionally active euchromatin.

These data are available at NCBI GEO, which also provides additional information about the experimental protocols.

## Methods

This track shows the combined results of three multiple biological replicates. For all arrays, the ChIP DNA was labeled with Cy5 and the control DNA was labeled with Cy3.

The data from replicates were quantile-normalized and median-scaled to each other (both Cy3 and Cy5 channels). Using a 1000 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 oligonucleotide positions with -log10(P-value) (>= 4), extending qualified positions upstream and downstream 250 bp, and requiring 1000 bp space between two sites. Top 400 sites are retained.

## 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

These data were generated and analyzed by the labs of Michael Snyder, Mark Gerstein and Sherman Weissman at Yale University.

## References

Cawley S, Bekiranov S, Ng HH, Kapranov P, Sekinger EA, Kampa D, Piccolboni A, Sementchenko V, Cheng J, Williams AJ et al. Unbiased mapping of transcription factor binding sites along human chromosomes 21 and 22 points to widespread regulation of noncoding RNAs. Cell 2004 Feb 20;116(4):499-509.

Euskirchen G, Royce TE, Bertone P, Martone R, Rinn JL, Nelson FK, Sayward F, Luscombe NM, Miller P, Gerstein M et al. CREB binds to multiple loci on human chromosome 22. Mol Cell Biol. 2004 May;24(9):3804-14.

Martone R, Euskirchen G, Bertone P, Hartman S, Royce TE, Luscombe NM, Rinn JL, Nelson FK, Miller P, Gerstein M et al. Distribution of NF-kappaB-binding sites across human chromosome 22. Proc Natl Acad Sci U S A. 2003 Oct 14;100(21):12247-52.

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