|
| Status |
Public on Dec 07, 2010 |
| Title |
AG174_HPUra_DnaA_rep3 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
AG174, HPUra treated, 30 minutes, DnaA IP
|
| Organism |
Bacillus subtilis |
| Characteristics |
strain: AG174
growth phase: exponential
isolation: anti-DnaA IP-associated DNA
antibody: anti-DnaA
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-DnaA was raised in chicken and comes from W. F. Burkholder (Stanford). The use of these antibodies is described in Goranov 2005 Proc Nat Acad Sci USA and Breier 2009 J. Bacteriol. Anti-DnaA serum came from chicken #1316. 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 |
628_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 |
|
