This track displays replication timing and was produced as part
of the ENCODE Project. Replication timing refers to the order in
which DNA is duplicated during the synthesis phase of the cell
cycle and is correlated with the expression of genes and the structure
of chromosomes. This track shows genome-wide assessment of DNA replication
timing in cell lines using NimbleGen tiling CGH microarrays. Each experiment
represents the relative enrichment of early versus late S-phase nascent strands
in a given cell line, with data represented as a loess-smoothed function
of individual timing values at probes spaced at even intervals across
the genome. Regions with high values indicate domains of early replication where
initiation occurs earlier in S-phase.
Display Conventions and Configuration
The graph displays a wavelet-smoothed signal of mean early/late S-phase ratios.
Metadata for a particular subtrack can be found by clicking the down arrow
in the list of subtracks.
Cells were grown according to the approved
ENCODE cell culture protocols.
Methods for replication timing profile creation and analysis are described in detail in
Hiratani et al. (2008) and Ryba et al. (June 2011).
Methods for individual stages of extraction, hybridization, scanning and
processing are summarized below.
For the extraction protocol, replication timing data were obtained by hybridizing
early and late replication intermediates to NimbleGen oligonucleotide arrays.
Replication intermediates were prepared from cells that were first pulse-labeled
with 5'-bromo-2'-deoxyuridine (BrdU) and then sorted into early and late stages
of S-phase by flow cytometry, followed by anti-BrdU immunoprecipitation of the
BrdU-substituted (nascent) replication intermediates newly synthesized either
early or late during S-phase. Samples were labeled after unbiased amplification
of recovered DNA by whole-genome amplification (WGA; Sigma, GenomePlex).
The hybridization set used the
NimbleGen standard hybridization protocol. Cy3- and Cy5-labeled DNA samples (6 µg each)
were co-hybridized to Nimblegen CGH arrays containing evenly-spaced oligonucleotide
probes across the human genome, with a median probe spacing of 1.1-5.8 kb.
No differences in smoothed data have been detected with probe densities from 100 bp to 5.8 kb. The NimbleGen MS 200 2 µm resolution scanner and GenePix software
were used per
NimbleGen's standard scanning protocol.
NimbleScan software was used to obtain .pair raw data per
manufacturer's instructions. Raw early/late data (i.e. from .pair files) from
two independent biological replicates, in which early- and late-replicating DNA
were labeled reciprocally, were loess-normalized to remove signal intensity-dependent bias.
The data were then scaled to a reference data set to have the same median absolute deviation
and then averaged (limma package, R/Bioconductor). The mean early/late ratios were
used to generate a final smoothed profile (i.e. processed data) using local polynomial
smoothing (loess, 300 kb span) for each chromosome using basic functions in
the statistical language R.
Technical data quality was assessed by verifying high autocorrelation between neighboring timing values.
Biological identity was confirmed by verifying consistent early or late replication by PCR at individual loci, as well as uniformity in replication profiles between replicate experiments.
This is Release 2 (July 2012) of this track. It adds 6 more data sets including additional replicates for
H1-hESC and H7-hESC and all new data for the iPS skin fibroblast bio samples.
These data were generated by the Florida State University ENCODE group.
David M. Gilbert
Hiratani I, Ryba T, Itoh M, Rathjen J, Kulik M, Papp B, Fussner E, Bazett-Jones DP, Plath K, Dalton S et al.
Genome-wide dynamics of replication timing revealed by in vitro models of mouse embryogenesis.
Genome Res. 2010 Feb;20(2):155-69.
Hiratani I, Ryba T, Itoh M, Yokochi T, Schwaiger M, Chang CW, Lyou Y, Townes TM, Schübeler D, Gilbert DM.
Global reorganization of replication domains during embryonic stem cell differentiation.
PLoS Biol. 2008 Oct 7;6(10):e245.
Pope BD, Tsumagari K, Battaglia D, Ryba T, Hiratani I, Ehrlich M, Gilbert DM.
DNA replication timing is maintained genome-wide in primary human myoblasts independent of D4Z4 contraction in FSH muscular dystrophy.
PLoS One. 2011;6(11):e27413.
Ryba T, Battaglia D, Pope BD, Hiratani I, Gilbert DM.
Genome-scale analysis of replication timing: from bench to bioinformatics.
Nat Protoc. 2011 Jun;6(6):870-95.
Ryba T, Hiratani I, Lu J, Itoh M, Kulik M, Zhang J, Schulz TC, Robins AJ, Dalton S, Gilbert DM.
Evolutionarily conserved replication timing profiles predict long-range chromatin interactions and distinguish closely related cell types.
Genome Res. 2010 Jun;20(6):761-70.
Ryba T, Hiratani I, Sasaki T, Battaglia D, Kulik M, Zhang J, Dalton S, Gilbert DM.
Replication timing: a fingerprint for cell identity and pluripotency.
PLoS Comput Biol. 2011 Oct;7(10):e1002225.
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