|
| Status |
Public on Dec 27, 2019 |
| Title |
MPBEpoD3 [re-analysis] |
| 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
|
| 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
|
| 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).
|
| Data processing |
Genome-wide RT profiles were constructed by Repli-chip, Repli-seq, Capture Repli-seq and deep whole genome sequencing (WGS). Replication timing profiles were scaled and pooled for analysis as previously described (Ryba et al., 2011; Marchal et al., 2018). Briefly, Log2 transformed early/late replication timing ratios were quantile normalized and loess smoothed in R
|
| |
|
| Submission date |
Apr 26, 2019 |
| Last update date |
Dec 27, 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 (2) |
| GSE130372 |
Replication timing alterations in leukemia reflect stable clinically-relevant changes in genome architecture [Repli-Chip] |
| GSE130374 |
Replication timing alterations in leukemia reflect stable clinically-relevant changes in genome architecture |
|
| Relations |
| Reanalysis of |
GSM1548634 |
