|
| Status |
Public on Mar 24, 2010 |
| Title |
Replication timing of iPS4, replicate 1 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Early-replicating DNA of iPS4
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: iPS4
cell type: iPSC (induced pluripotent stem cells)
|
| 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
|
| 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 iPS4
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: iPS4
cell type: iPSC (induced pluripotent stem cells)
|
| 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
|
| 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 (34 ug each) were co-hybridized to Nimblegen CGH arrays containing one oligonucleotide probe every 1.1 kb across the human genome.
|
| 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 |
submitted as of 1/20/10
|
| 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 |
Jan 25, 2010 |
| Last update date |
Mar 24, 2010 |
| Contact name |
Tyrone Ryba |
| Organization name |
Florida State University
|
| Department |
Biological Science
|
| Lab |
David Gilbert
|
| Street address |
319 Stadium Dr.
|
| City |
Tallahassee |
| State/province |
FL |
| ZIP/Postal code |
32306 |
| Country |
USA |
| |
|
| Platform ID |
GPL9973 |
| Series (2) |
| GSE20027 |
Evolutionarily conserved replication timing profiles distinguish cell types and predict long range chromatin interaction |
| GSE51334 |
DNA replication-timing boundaries separate stable chromosome domains with cell-type-specific functions |
|
