This annotation shows smoothed replication timing for DNA synthesis as the time of 50% replication (TR50).
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The experimental strategy adopted to map this profile involved isolation of replication products from HeLa cells synchronized at the G1-S boundary by thymidine-aphidicolin double block. Cells released from the block were labeled with BrdU at every two-hour interval of the 10 hours of S-phase and DNA was isolated from them. The heavy-light (H/L) DNA representing the pool of DNA replicated during each two-hour labeling period was separated from the unlabeled DNA by double cesium chloride density gradient centrifugation. The purified H/L DNA was then hybridized to a high-density genome-tiling Affymetrix array comprised of all unique probes within the ENCODE regions.
The time of replication of 50% (TR50) of each microarray probe was calculated by accumulating the sum over the five time points and linearly interpolating the time when 50% was reached. Each probe was also classified as temporally specific or non-specific based on whether at least 50% of the accumulated signal appeared in a single time point or not.
The TR50 data for all specific probes were then lowess-smoothed within a 60 kb window to provide the profile displayed in the annotation.
The replication experiments were completed for two biological sets in the HeLa adherent cell line.
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.
Jeon, Y., Bekiranov, S., Karnani, N., Kapranov, P., Ghosh, S., MacAlpine, D., Lee, C., Hwang, D.S., Gingeras, T.R. and Dutta, A. Temporal profile of replication of human chromosomes. Proc Natl Acad Sci U S A 102(18), 6419-24 (2005).