|
| Status |
Public on Nov 25, 2009 |
| Title |
Replication timing of 46C ESC |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Early-replicating DNA of 46C ESC
|
| Organism |
Mus musculus |
| Characteristics |
cell line: 46C
developmental stage: ESC
|
| 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.
|
| |
|
| Channel 2 |
| Source name |
Late-replicating DNA of 46C ESC
|
| Organism |
Mus musculus |
| Characteristics |
cell line: 46C
developmental stage: ESC
|
| 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].
|
| |
|
| 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 |
|
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