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Sample GSM1215792 Query DataSets for GSM1215792
Status Public on May 29, 2014
Title Replication timing of TS cells, replicate 3 (TSC 4)
Sample type genomic
 
Channel 1
Source name Early-replicating DNA of TSCs
Organism Mus musculus
Characteristics Stage: Early-replicating DNA of TSCs
Growth protocol Mouse TSCs were obtained from Dr. Janet Rossant, Hospital for Sick Children, (Toronto, Canada) and maintained in DMEM/F12 with 15 mM Hepes, 20% FBS, 2 mM glutamine, 100 ug/ml streptomycin, 1 mM sodium pyruvate, 100 uM BME, supplemented with Activin, FGF4, and Heparin following (Erlebacher et al, Developmental Biology 2004). Trophoblast stem cells were differentiated into trophoblast giant cells by replacing the FGF, Activin and Heparin in the media with retinoic acid (Yan et al., 2001; Erlebacher et al., 2004). Mature TGCs are seen after 4-6 days of differentiation (Carney et al., 1993). Cells were trypsinized for 4 minutes before the extract protocol.
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 TSCs
Organism Mus musculus
Characteristics Stage: Late-replicating DNA of TSCs
Growth protocol Mouse TSCs were obtained from Dr. Janet Rossant, Hospital for Sick Children, (Toronto, Canada) and maintained in DMEM/F12 with 15 mM Hepes, 20% FBS, 2 mM glutamine, 100 ug/ml streptomycin, 1 mM sodium pyruvate, 100 uM BME, supplemented with Activin, FGF4, and Heparin following (Erlebacher et al, Developmental Biology 2004). Trophoblast stem cells were differentiated into trophoblast giant cells by replacing the FGF, Activin and Heparin in the media with retinoic acid (Yan et al., 2001; Erlebacher et al., 2004). Mature TGCs are seen after 4-6 days of differentiation (Carney et al., 1993). Cells were trypsinized for 4 minutes before the extract protocol.
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 (31 ug each) were co-hybridized to Nimblegen CGH arrays containing one oligonucleotide probe every 3.5 kb across the entire mouse 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 NimbleScan software was used to obtain .pair raw data per manufacturer's instructions. Raw early/late data (i.e. from .pair files) from three independent biological replicates in which early and late replicating DNA were labeled 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 Aug 26, 2013
Last update date May 30, 2014
Contact name Roberta L Hannibal
E-mail(s) robertah@stanford.edu
Organization name Stanford University
Department Genetics
Street address 300 Pasteur Drive
City Stanford
State/province CA
ZIP/Postal code 94305
Country USA
 
Platform ID GPL15225
Series (2)
GSE50207 Trophoblast giant cell underrepresentation (tiling)
GSE50585 Trophoblast giant cell underrepresentation

Data table header descriptions
ID_REF
VALUE loess normalized early/late

Data table
ID_REF VALUE
CHR01FS003002567 -0.575096815
CHR01FS003003786 -0.579659132
CHR01FS003011391 -0.607130737
CHR01FS003014357 -0.617381387
CHR01FS003018614 -0.631638986
CHR01FS003022241 -0.643363404
CHR01FS003026285 -0.655976462
CHR01FS003034358 -0.679706355
CHR01FS003037908 -0.689529456
CHR01FS003040093 -0.695389584
CHR01FS003041991 -0.700364962
CHR01FS003047748 -0.714801864
CHR01FS003050305 -0.720898272
CHR01FS003052595 -0.72618921
CHR01FS003057911 -0.737874531
CHR01FS003061122 -0.744561372
CHR01FS003065156 -0.752606453
CHR01FS003068324 -0.758673219
CHR01FS003072431 -0.766246727
CHR01FS003076180 -0.772908844

Total number of rows: 707639

Table truncated, full table size 20278 Kbytes.




Supplementary file Size Download File type/resource
GSM1215792_544770_Cycle2_2013-04-30_TSC4_532.pair.gz 12.5 Mb (ftp)(http) PAIR
GSM1215792_544770_Cycle2_2013-04-30_TSC4_635.pair.gz 12.5 Mb (ftp)(http) PAIR
Processed data included within Sample table

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