|
| Status |
Public on May 28, 2015 |
| Title |
MPB_EpoD3_R1 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Early-replicating DNA
|
| Organism |
Homo sapiens |
| Characteristics |
differentiation stage: Erythroid differentiation day 3 from CD34+ peripheral blood stem and progenitor cells
cell line: Primary cells
|
| Growth protocol |
Erythroid differentiation was induced in the CD45RA- fraction of cells (from human CD34+ peripheral blood stem cells) by an erythroid “cocktail” supplementing the stem cell expansion medium with 10ng/ml SCF, 5ng/ml IL3, recombinant erythropoietin 1 unit/ml, dexamethasone with a final concentration of 2 micromolar, and Estradiol with a final concentration of 0.2 micromolar for 3 days (Migliaccio et al. 2009. Curr Opin Hematol 16: 259–268; Mahajan et al. 2009, Exp Hematol 37: 1143–1156.e3).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Replication timing data were obtained by hybridizing early and late replication intermediates to NimbleGen oligonucleotide arrays, as previously described (Ryba, T. et al., 2011. Nat Protoc 6: 870–895). Briefly, replication intermediates were prepared from cells 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] and Ryba, T. et al [Nat Protoc (2011) 6: 870–895].
|
| 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
|
| Organism |
Homo sapiens |
| Characteristics |
differentiation stage: Erythroid differentiation day 3 from CD34+ peripheral blood stem and progenitor cells
cell line: Primary cells
|
| Growth protocol |
Erythroid differentiation was induced in the CD45RA- fraction of cells (from human CD34+ peripheral blood stem cells) by an erythroid “cocktail” supplementing the stem cell expansion medium with 10ng/ml SCF, 5ng/ml IL3, recombinant erythropoietin 1 unit/ml, dexamethasone with a final concentration of 2 micromolar, and Estradiol with a final concentration of 0.2 micromolar for 3 days (Migliaccio et al. 2009. Curr Opin Hematol 16: 259–268; Mahajan et al. 2009, Exp Hematol 37: 1143–1156.e3).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Replication timing data were obtained by hybridizing early and late replication intermediates to NimbleGen oligonucleotide arrays, as previously described (Ryba, T. et al., 2011. Nat Protoc 6: 870–895). Briefly, replication intermediates were prepared from cells 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] and Ryba, T. et al [Nat Protoc (2011) 6: 870–895].
|
| 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. Briefly, Cy3 and Cy5 labeled DNA samples (31 ug each) were co-hybridized to Nimblegen CGH arrays containing oligonucleotide probes across the entire 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 |
Human genomic data
|
| Data processing |
DEVA (Nimblegen) software was used to obtain .pair raw data per manufacturer's instructions. Raw early/late data (i.e. from .pair files) from independent biological replicates in which early and late replicating DNA were labeled reciprocally were loess-normalized to remove signal intensity-dependent bias (limma package, R/Bioconductor) and 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 |
Nov 18, 2014 |
| Last update date |
Apr 26, 2019 |
| Contact name |
David M Gilbert |
| Organization name |
Florida State University
|
| Department |
Biology
|
| Lab |
Gilbert
|
| Street address |
319 Stadium Drive
|
| City |
Tallahassee |
| State/province |
FL |
| ZIP/Postal code |
32306-4295 |
| Country |
USA |
| |
|
| Platform ID |
GPL14965 |
| Series (1) |
| GSE63428 |
Distinct classes of genes behave as either drivers or dependents of replication timing switches (Replication-Timing). |
|
| Relations |
| Reanalyzed by |
GSM3737002 |
