|
| Status |
Public on Apr 06, 2010 |
| Title |
stationary phase broth culture rep 3 |
| Sample type |
RNA |
| |
|
| Source name |
stationary phase broth culture
|
| Organism |
Yersinia pestis |
| Characteristics |
strain: KIM6+
|
| Treatment protocol |
RNA isolation from cultures grown to early stationary growth phase (7 h).
|
| Growth protocol |
The bacterial strain used in this study was Y. pestis KIM6+, which lacks the 70-kb virulence plasmid that is not required for flea infection or blockage. A KIM6+ strain deleted of yitR (y0181) was produced by allelic exchange, using the pCVD442 suicide vector system (11). This mutant was complemented by electroporation with a recombinant pWKS130 plasmid containing the wild-type yitR promoter and orf. The ΔyitR mutant was also transformed with pWKS130 alone to generate an empty vector control strain. For in vitro planktonic samples, bacteria were grown from frozen stocks in brain heart infusion (BHI) medium at 28°C, followed by two successive transfers in Luria Bertani broth supplemented with 100 mM MOPS, pH 7.4 (LB/MOPS) at 21°C. An inoculum of 10^4 cells/ml was added to 50 ml of LB/MOPS and incubated at 21°C with shaking at 250 rpm until exponential (OD600 = 2.5) or stationary phase (OD600 = 4.5).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Approximately 0.5 ml of the exponential phase culture and 0.25 ml of the stationary phase culture was resuspended in 1ml and 0.5 ml, respectively, of RNAprotect bacterial reagent (Qiagen; Valencia, CA), incubated for 5 min at room temperature, and centrifuged at 21°C for 5 min prior to RNA extraction. RNA was isolated from six independent samples from in vitro and flow cell cultures and two independent samples from pooled blocked fleas (Fig. S1) using the RNeasy Mini Kit (Qiagen). Flea-derived RNA samples were secondarily split into three technical replicates each. RNA integrity was verified on a Bioanalyzer 2100 (Agilent Technologies; Santa Clara, CA).
|
| Label |
biotin
|
| Label protocol |
Total RNA (100 ng) was amplified and labeled with Modified biotin-11-CTP (Perkin Elmer; Waltham, MA) and biotin-16-UTP (Roche Molecular Biochemicals, Pleasanton, CA) by using the Message-Amp II-Bacteria amplified antisense RNA (aRNA) kit (Ambion; Austin, TX). Amplified RNA was then fragmented using Ambion’s Fragmentation reagent (Applied Biosystems), hybridized to the RML custom Affymetrix GeneChip.
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| |
|
| Hybridization protocol |
All samples were hybridized for 16 hr at 45C on our custom Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 450.
|
| Scan protocol |
GeneChips were scanned using the Affymetrix GeneChip Scanner 7GPlus.
|
| Description |
Gene expression data from_stationary phase broth culture rep 3
|
| Data processing |
The data were analyzed with GCOS 1.4 using Affymetrix default analysis with a scale filter for Yp genes setting and global scaling as normalization method. The trimmed mean target intensity of each array was arbitrarily set to 500.
|
| |
|
| Submission date |
Jun 08, 2009 |
| Last update date |
Apr 06, 2010 |
| Contact name |
Dan Sturdevant |
| E-mail(s) |
dsturdevant@niaid.nih.gov
|
| Phone |
4063639248
|
| Organization name |
NIH
|
| Department |
NIAID
|
| Lab |
RTS
|
| Street address |
903 S 4th street
|
| City |
Hamilton |
| State/province |
MT |
| ZIP/Postal code |
59840 |
| Country |
USA |
| |
|
| Platform ID |
GPL2129 |
| Series (1) |
| GSE16493 |
Preadaptation of Yersinia pestis to resist mammalian innate immunity during transit through the flea vector |
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