ReplDB Database - Help and documentation

Replication Timing Analysis

Replication timing data were obtained by hybridizing early and late replication intermediates to Nimblegen oligonucleotide arrays, as described in Hiratani et. al. (PloS Biology, in press; see "Cite Us"). Briefly, replication intermediates are prepared from cells that are first pulse-labeled with BrdU and then sorted into early and late 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. After unbiased amplification of recovered DNA, the samples are differentially labeled with Cy3 and Cy5 and hybridized to Nimblegen CGH arrays containing one oligonucleotide probe every 5-6 kb across the mouse genome (Nimblegen, 2006-07-26_MM8_WG_CGH). Raw data from two independent biological replicates in which the early and late replicating DNA were labeled reciprocally with Cy3 and Cy 5 (dye swap) are loess-normalized and scaled to have the same median-absolute deviation using the limma package (R/Bioconductor) and then averaged. Finally, the data are smoothed with a weighted moving average (loess: local polynomial smoothing). Additional quality controls can be found in Hiratani et. al. (PloS Biology, in press; see "Cite Us").

Gene Expression Analysis (Steady-State Transcript Levels)

This is standard Affymetrix GeneChip analysis of steady state transcript levels from total RNA, performed in triplicate from cells grown under identical conditions to those used to evaluate replication timing (GeneChip MOE 430 analyzing 45,037 single-copy murine genes and EST clusters). We matched these data to 15,143 mouse mm8 RefSeq genes (which correspond to 81% of 18,702 unique RefSeq genes). Each data set was first subject to Affymetrix Quality Control measures using GeneChip® Operating Software (GCOS) according to manufacturer’s protocols. All three data sets passed this quality control test and were subjected to the probe logarithmic intensity error (PLIER) method developed by Affymetrix for calculating probe signals. The three data sets were then averaged. As for replication timing, additional quality controls can be found in Hiratani et. al. (TBA when accepted).

Definitions of Data Sets

Data sets are defined by a combination of five entries, as described below.

• Build - This indicates the version of the genomic sequence information that was used to assemble the microarray chip in the particular experiment. Builds change slightly as sequence information becomes updated, so the exact base pair position of any given DNA sequence will change as the sequence information becomes annotated. The build information indicated in each data set shows the build used for chromosomal coordinates of probes on the particular array type used.
   
• Data Type - Indicates the property being measured in the indicated experiment. At present, replication timing and transcription data are shown. In the future, data for other genome-wide properties of chromosomes may be displayed.
   
• Cell line - This indicates the name of cell lines employed. References for these cells lines are as follows:
   

  Mouse Embryonic Stem Cell line D3

  Doetschman TC, Eistetter H, Katz M, Schmidt W, Kemler R: The in vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium. J Embryol Exp Morphol 1985, 87:27-45
   

  Mouse Embryonic Stem Cell line 46C

  Ying QL, Stavridis M, Griffiths D, Li M, Smith A: Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture. Nat Biotechnol 2003, 21(2):183-186.
   

  Mouse Embryonic Stem Cell line TT2

  Yagi T, Tokunaga T, Furuta Y, Nada S, Yoshida M, Tsukada T, Saga Y, Takeda N, Ikawa Y, Aizawa S: A novel ES cell line, TT2, with high germline-differentiating potency. Anal Biochem 1993, 214(1):70-76
   

• Differentiation State – This is the tissue or tissue-type represented by the cell line used and grown under the indicated conditions. Currently, there are four such differentiation states:
   

  ES

  Undifferentiated ES cells
   

  NPC/EBM9

  The 9th day of differentiation following an established neural differentiation protocol that differentiates embryoid bodies to Sox1 positive NPCs in conditioned medium.
  
  Reference: Rathjen J, Haines BP, Hudson KM, Nesci A, Dunn S, Rathjen PD. Directed differentiation of pluripotent cells to neural lineages: homogeneous formation and differentiation of a neurectoderm population. Development. 2002 Jun;129(11):2649-61.
   

  NPC/ASd6

  The 6th day of differentiation following an established neural differentiation protocol that differentiates ES cells to Sox1 positive NPCs in monolayer cultures using defined medium.
  
  Reference: Ying QL, Stavridis M, Griffiths D, Li M, Smith A: Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture. Nat Biotechnol 2003, 21(2):183-186.
   

  iPS

  "Induced Pluripotent Cells" re-programmed from tail-tip fibroblasts derived from a 129xBL-6 hybrid strain of mice to the pluripotent state as described.
  
  Reference: Hanna J, Wernig M, Markoulaki S, Sun CW, Meissner A, Cassady JP, Beard C, Brambrink T, Wu LC, Townes TM et al: Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin. Science 2007, 318(5858):1920-1923.
   

  Reference:
  Rathjen J, Haines BP, Hudson KM, Nesci A, Dunn S, Rathjen PD. Directed differentiation of pluripotent cells to neural lineages: homogeneous formation and differentiation of a neurectoderm population. Development. 2002 Jun;129(11):2649-61.
   
• Chip ID – This is the unique identifier for each data set. One Chip ID represents the data for one experiment (e.g. one microarray hybridization). At present, only averages of multiple replicates are displayed, indicated by combining all Chip ID numbers into one larger number. This number itself is not useful except to communicate comments regarding a particular experiment. However, from the viewer display, the Chip ID number for each data set is set up as a link to the relevant "Data Set Details" (also accessible from the "Database" menu item), which provides additional details of each data set. The Chip ID is also useful for downloading the entire data set within the "Download Data" menu item.