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Sample GSM580941 Query DataSets for GSM580941
Status Public on Dec 07, 2010
Title AG174_HPUra_DnaC_rep1
Sample type genomic
 
Channel 1
Source name AG174, HPUra treated, 30 minutes, DnaC IP
Organism Bacillus subtilis
Characteristics strain: AG174
growth phase: exponential
isolation: anti-DnaC IP-associated DNA
antibody: anti-DnaC
treatment: 30 minutes HPUra
Treatment protocol To induce replication stress by arresting the replicative polymerase PolC, the inhibitor HPUra was added at a final concentration of 38 ug/mL to exponentially growing cells for 30 minutes.
Growth protocol Cells were grown in minimal 1% glucose medium until OD600 reached approximately 0.6. At this point, cells were treated with HPUra or not, and growth was continued for another 30 minutes before harvesting.
Extracted molecule genomic DNA
Extraction protocol Chromatin immunoprecipitation (ChIP) of DNA bound to native proteins was done essentially as described (1998, Lin and Grossman, Cell, 92:675-685 [PMID 9506522]; 2007, Breier and Grossman, Molecular Microbiology, 64:703-718 [PMID 17462018]).

Anti-DnaB/C/D were custom antibodies (sera), and were obtained from Covance (CRP, Inc); they do not have a catalog number. Anti-DnaB, anti-DnaC and anti-DnaD were raised in rabbits after purification of tagged proteins in the Grossman lab. For the experiments, the following bleeds were used:
DnaB: rabbit HM654, exsanguination
DnaC: rabbit HM6351, test bleed 2
DnaD: rabbit HM6354, production bleed 3
The IP requires a second antibody: AffiniPure Donkey Anti-Chicken IgY from Jackson ImmunoResearch Laboratories Inc. Catalog #703-005-155.
Label Cy5
Label protocol Samples were prepared for microarray analysis by labelling aliquots of the resuspended immunoprecipitated DNA or total control DNA with aminoallyl-dUTP using 13 U Sequenase (USB) and 5 μg random nonamer. The labelled samples were purified, conjugated to Cy3 or Cy5, and hybridized to microarrays.
 
Channel 2
Source name AG174, HPUra treated, 30 minutes, total DNA
Organism Bacillus subtilis
Characteristics strain: AG174
growth phase: exponential
treatment: 30 minutes HPUra
isolation: total chromosomal DNA
antibody: none
Treatment protocol To induce replication stress by arresting the replicative polymerase PolC, the inhibitor HPUra was added at a final concentration of 38 ug/mL to exponentially growing cells for 30 minutes.
Growth protocol Cells were grown in minimal 1% glucose medium until OD600 reached approximately 0.6. At this point, cells were treated with HPUra or not, and growth was continued for another 30 minutes before harvesting.
Extracted molecule genomic DNA
Extraction protocol Chromatin immunoprecipitation (ChIP) of DNA bound to native proteins was done essentially as described (1998, Lin and Grossman, Cell, 92:675-685 [PMID 9506522]; 2007, Breier and Grossman, Molecular Microbiology, 64:703-718 [PMID 17462018]).
Label Cy3
Label protocol Samples were prepared for microarray analysis by labelling aliquots of the resuspended immunoprecipitated DNA or total control DNA with aminoallyl-dUTP using 13 U Sequenase (USB) and 5 μg random nonamer. The labelled samples were purified, conjugated to Cy3 or Cy5, and hybridized to microarrays.
 
 
Hybridization protocol Samples were hybridized as described in Auchtung et al. 2005 PNAS 102:12554-9 [PMID 16105942].
Scan protocol Arrays were scanned and analyzed with GenePix Pro 3.0 (Axon Instruments).
Description 636_bottom
Data processing GPR files were loaded and processed in Prep+07 (BMC Bioinformatics 2009, 10:16 [PMID 19134227; doi:10.1186/1471-2105-10-16). Lowess normalization was performed and spots for which signal was 90% within 2SD above background were removed. Results from the Prep+07 analysis were manually curated in Microsoft Excel for spots that had <2 datapoints with quality data for downstream analysis.
 
Submission date Aug 18, 2010
Last update date Sep 16, 2011
Contact name Alan D. Grossman
E-mail(s) clee2@mit.edu, adg@mit.edu
Phone 617-253-6702
Organization name MIT
Department Biology
Lab Grossman
Street address 31 Ames Street, 68-530
City Cambridge
State/province MA
ZIP/Postal code 02142
Country USA
 
Platform ID GPL10707
Series (1)
GSE23686 Genome-wide binding of replication initiation proteins in Bacillus subtilis

Data table header descriptions
ID_REF
VALUE Lowess-normalized log2 ratio (IP/total)

Data table
ID_REF VALUE
1 null
2 -0.282375
3 -0.282525
4 -0.24865
5 -0.126105
6 0.132508
7 -0.03322
8 0.169019
9 null
10 -0.0832226
11 -0.288272
12 -0.30126
13 0.299592
14 -0.150751
15 -0.0889665
16 0.059865
17 null
18 -0.140245
19 0.017267
20 -0.0558409

Total number of rows: 4624

Table truncated, full table size 63 Kbytes.




Supplementary file Size Download File type/resource
GSM580941.gpr.gz 359.4 Kb (ftp)(http) GPR
Processed data included within Sample table

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