Temporal profiling of DNA replication in human ENCODE regions is displayed as five tracks (0-2, 2-4, 4-6, 6-8, 8-10), corresponding to two-hour intervals of S phase.
The experimental strategy adopted to map this profile involves isolation of replication products from HeLa cells synchronized at the G1-S boundary by thymidine-aphidicolin double block and released from the block followed by BrdU labeling at every two-hour interval of the 10 hrs of S phase. The heavy-light DNA representing the pool of DNA replicated during each two-hour labeling period was purified by double cesium chloride density gradient centrifugation and hybridized to high-density genome-tiling Affymetrix array comprised of all unique probes within the ENCODE regions.
The raw data generated through the microarray experiments was processed by computing the enrichment of signal in a particular part of S-phase relative to the entirety of S-phase (10hrs). High confidence (p-value = 1E5) regions of replication were mapped by applying the Wilcoxon Rank Sum test in a sliding window of size 10 kb using the standard Affymetrix data analyses tools and the April 2003 version of human genome assembly. These coordinates were then mapped to the July 2003 assembly by UCSC, using the liftOver tool.
The criteria for data verification involve two biological and three technical replicates. The submitted data is from one biological experimental set. The tracks will be updated as soon as the results from the second biological replicate have been analyzed.
Data generation and analysis for this track were performed by the DNA replication group in the Dutta Lab at the University of Virginia: Neerja Karnani, Christopher Taylor, Hakkyun Kim, Louis Lim, Ankit Malhotra, Gabe Robins and Anindya Dutta.
Neerja Karnani and Christopher Taylor prepared the data for presentation in the UCSC Genome Browser.