Description

This track shows the location of sites showing transcription for all ENCODE regions in several cell types, using Affymetrix arrays. Retinoic acid-stimulated HL-60 cells were harvested after 0, 2, 8, and 32 hours. Purified cytosolic polyA+ RNA from unstimulated GM06990 and HeLa cells, as well as purified polyA+ RNA from the RA-stimulated HL-60 samples, was hybridized to Affymetrix ENCODE oligonucleotide tiling arrays, which have 25-mer probes tiled every 22 bp on average in the non-repetitive ENCODE regions. Clustered sites are shown in separate subtracks for each cell type and for each of the four timepoints for RA-stimulated HL-60.

Data for all biological replicates can be downloaded from Affymetrix in wiggle, cel, and soft formats.

Display Conventions and Configuration

To show only selected subtracks, uncheck the boxes next to the tracks that you wish to hide.

Color differences among the subtracks are arbitrary. They provide a visual cue for distinguishing between the different cell types and timepoints.

Methods

The data from replicate arrays were quantile-normalized (Bolstad et al., 2003) and all arrays were scaled to a median array intensity of 22. Within a sliding 101 bp window centered on each probe, an estimate of RNA abundance (signal) was found by calculating the median of all pairwise average PM-MM values, where PM is a perfect match and MM is a mismatch. Both Kapranov et al. (2002) and Cawley et al. (2004) are good references for the experimental methods; Cawley et al. also describes the analytical methods.

Verification

Three independent biological replicates were generated and hybridized to duplicate arrays (two technical replicates). Transcribed regions (see the Affy RNA Signal track) were generated from the composite signal track by merging genomic positions to which probes are mapped. This merging was based on a 5% false positive rate cutoff in negative bacterial controls, a maximum gap (MaxGap) of 50 base-pairs and minimum run (MinRun) of 40 base-pairs. A random subset of transfrags were verified by RACE where the RACE primers were designed based on the sequences of the transfrags.

Credits

These data were generated and analyzed by the Gingeras/Struhl collaboration with the Tom Gingeras group at Affymetrix and the Kevin Struhl group at Harvard Medical School.

References

Please see the Affymetrix Transcriptome site for a project overview and additional references to Affymetrix tiling array publications.

Bolstad, B. M., Irizarry, R. A., Astrand, M., and Speed, T. P. A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinformatics 19(2), 185-193 (2003).

Cawley, S., Bekiranov, S., Ng, H. H., Kapranov, P., Sekinger, E. A., Kampa, D., Piccolboni, A., Sementchenko, V., Cheng, J., Williams, A. 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).

Kapranov, P., Cawley, S. E., Drenkow, J., Bekiranov, S., Strausberg, R. L., Fodor, S. P., and Gingeras, T. R. Large-scale transcriptional activity in chromosomes 21 and 22. Science 296(5569), 916-919 (2002).