|
Status |
Public on Nov 25, 2009 |
Title |
Replication timing of D3 EBM3 |
Sample type |
genomic |
|
|
Channel 1 |
Source name |
Early-replicating DNA of EBM3
|
Organism |
Mus musculus |
Characteristics |
cell line: D3 developmental stage: EBM3 (day3 differentiated ESCs)
|
Extracted molecule |
genomic DNA |
Extraction protocol |
Replication timing data were obtained by hybridizing early and late replication intermediates to NimbleGen oligonucleotide arrays, as described in Hiratani et al [PLoS Biology (2008) 6: e245]. Briefly, replication intermediates are prepared from cells that are first pulse-labeled with BrdU and then sorted into early (1st half of S) and late (2nd half of S) stages of S-phase by flow cytometry, followed by anti-BrdU immunoprecipitation of the BrdU-substituted (nascent) replication intermediates that were synthesized either early or late during S-phase. Samples were labeled after unbiased amplification of recovered DNA by whole-genome amplification (WGA; Sigma, GenomePlex). Further details can be found in Hiratani et al [PLoS Biology (2008) 6: e245].
|
Label |
Cy3,Cy5
|
Label protocol |
Standard NimbleGen protocol (www.nimblegen.com/products/lit/index.html). Briefly, randomly-amplified BrdU-immunoprecipitated DNA samples (1 ug each) were Cy3- or Cy5-labeled by Klenow reaction using end-labeled Cy3- or Cy5-random 9-mers.
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|
|
Channel 2 |
Source name |
Late-replicating DNA of EBM3
|
Organism |
Mus musculus |
Characteristics |
cell line: D3 developmental stage: EBM3 (day3 differentiated ESCs)
|
Extracted molecule |
genomic DNA |
Extraction protocol |
Replication timing data were obtained by hybridizing early and late replication intermediates to NimbleGen oligonucleotide arrays, as described in Hiratani et al [PLoS Biology (2008) 6: e245]. Briefly, replication intermediates are prepared from cells that are first pulse-labeled with BrdU and then sorted into early (1st half of S) and late (2nd half of S) stages of S-phase by flow cytometry, followed by anti-BrdU immunoprecipitation of the BrdU-substituted (nascent) replication intermediates that were synthesized either early or late during S-phase. Samples were labeled after unbiased amplification of recovered DNA by whole-genome amplification (WGA; Sigma, GenomePlex). Further details can be found in Hiratani et al [PLoS Biology (2008) 6: e245].
|
Label |
Cy5,Cy3
|
Label protocol |
Standard NimbleGen protocol (www.nimblegen.com/products/lit/index.html). Briefly, randomly-amplified BrdU-immunoprecipitated DNA samples (1 ug each) were Cy3- or Cy5-labeled by Klenow reaction using end-labeled Cy3- or Cy5-random 9-mers.
|
|
|
|
Hybridization protocol |
Standard NimbleGen protocol (www.nimblegen.com/products/lit/index.html). Briefly, Cy3 and Cy5 labeled DNA samples (6 ug each) were co-hybridized to Nimblegen CGH arrays containing one oligonucleotide probe every 5.8 kb across the mouse genome (Nimblegen, 2006-07-26_MM8_WG_CGH).
|
Scan protocol |
GenePix 4000B scanner (Molecular Devices) and GenePix software were used per NimbleGen's standard protocol (www.nimblegen.com/products/lit/index.html).
|
Description |
merge replicates 1 and 2
|
Data processing |
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, scaled to a reference data set to have the same median absolute deviation and averaged (limma package, R/Bioconductor).The mean early/late ratios were used to generate a 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. Processed data sets can be graphically displayed (and are also downloadable) on our web site (http://www.replicationdomain.org). Further details can be found in Hiratani et al [PLoS Biology (2008) 6: e245].
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|
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Submission date |
Sep 04, 2009 |
Last update date |
Apr 16, 2013 |
Contact name |
David M. Gilbert |
E-mail(s) |
gilbert@bio.fsu.edu
|
Phone |
8506457583
|
Organization name |
Florida State University
|
Street address |
319 Stadium Drive
|
City |
Tallahassee |
State/province |
Florida |
ZIP/Postal code |
32306-4295 |
Country |
USA |
|
|
Platform ID |
GPL9156 |
Series (4)
|
GSE17983 |
Replication Timing Reveal An Epigenetic Commitment To Differentiation Prior To Germ Layer Specification (WG_CGH, RT) |
GSE18019 |
Genome-Wide Dynamics of Replication Timing Revealed by In Vitro Models of Mouse Embryogenesis |
GSE49847 |
A comparative encyclopedia of DNA elements in the mouse genome |
GSE51334 |
DNA replication-timing boundaries separate stable chromosome domains with cell-type-specific functions |
|