|
| Status |
Public on Jun 05, 2012 |
| Title |
Replication timing of Hsa21 in IMR90 fibroblasts, average of 2 biological replicates |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Early-replicating DNA of IMR90 fibroblasts
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: IMR90 fibroblasts
dna replication status: Early-replicating DNA
|
| 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]. For complete extraction protocol, please refer to PubMed ID: 21637205.
|
| 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.
|
| |
|
| Channel 2 |
| Source name |
Late-replicating DNA of IMR90 fibroblasts
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: IMR90 fibroblasts
dna replication status: Late-replicating DNA
|
| 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]. For complete extraction protocol, please refer to PubMed ID: 21637205.
|
| 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.
|
| |
|
| |
| 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 70 bp across human chromosome 21.
|
| 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 |
Hsa21 data
Two biological replicates: 348297_IMR901_635.pair and 348297_IMR902_532.pair raw data files represent the Early-replicating DNA, 348297_IMR901_532.pair and 348297_IMR902_635.pair represent the Late-replicating DNA
|
| 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. For Tc1-21 datasets, early/late data was arranged according to the Tc1-21 sequence (Sequencing data for Tc1-21 is deposited in the ENA database, Study Accession number: ERP000439) prior to smoothing. 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].
In this study we analyzed a mouse strain (Tc1) that contains a rearranged human chromosome 21 (Tc1-21). To make sense of Tc1-21 data from a human chromosome 21 array, we reordered the array probes to reflect Tc1-21's rearranged sequence and then processed the data. So some array probes are missing from the processed data file (correspond to deleted regions) while others are found twice (correspond to duplicated regions). The duplicated regions are present in the supplementary processed data (.txt) files on each Sample record.
HSA21_START = Start position of oligo in human chromosome 21 (Hg18, NCBI 36)
HSA21_END = End position of oligo in human chromosome 21 (Hg18, NCBI 36)
Tc1.21_START = Start position of oligo in Tc1 trans-chromosome 21 (Sequencing data for Tc1-21 is deposited in the ENA database, Study Accession number: ERP000439)
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| |
|
| Submission date |
Jun 04, 2012 |
| Last update date |
Jun 05, 2012 |
| Contact name |
Benjamin Pope |
| Organization name |
Florida State University
|
| Department |
Biological Science
|
| Lab |
David Gilbert
|
| Street address |
319 Stadium Dr
|
| City |
Tallahassee |
| State/province |
FL |
| ZIP/Postal code |
32306-4295 |
| Country |
USA |
| |
|
| Platform ID |
GPL15656 |
| Series (2) |
| GSE38472 |
Replication-Timing Boundaries Facilitate Cell-type and Species-specific Regulation of a Rearranged Human Chromosome in Mouse |
| GSE51334 |
DNA replication-timing boundaries separate stable chromosome domains with cell-type-specific functions |
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