|
| Status |
Public on Dec 07, 2010 |
| Title |
AG174_HPUra_DnaB_rep1 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
AG174, HPUra treated, 30 minutes, DnaB IP
|
| Organism |
Bacillus subtilis |
| Characteristics |
strain: AG174
growth phase: exponential
isolation: anti-DnaB IP-associated DNA
antibody: anti-DnaB
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 |
633_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 |
|
