|
| Status |
Public on Sep 06, 2012 |
| Title |
LbrAdeletionMutant vs ScottA rep1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
LbrAdeletion mutant
|
| Organism |
Listeria monocytogenes |
| Characteristics |
strain background: ScottA
genotype/variation: LbrA deletion
|
| Treatment protocol |
No treatment was applied before extract preparation.
|
| Growth protocol |
Frozen L. monocytogenes cultures were activated by incubating in trypticase soy broth supplemented with 0.5% yeast extract (TSBYE) at 37°C for 18 h, and transferred at least once prior to assessments.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using the RNeasy mini kit (Qiagen, Valencia, CA) following manufacturer’s procedure with slight modification. Briefly, 23.75 ml of fresh TSBYE was inoculated with overnight cultures at 2.5% and grown at 37°C for 6 h until reached at OD600 of 0.6 – 0.7. One mL of aliquots were removed and placed in 4°C refrigerator for 15 min, and the cells were collected by centrifugation at 8000xg for 5 min. The cells were resuspended in 100μL of TE buffer and 6μL of lysozyme (50μg/mL) followed by incubation at 30°C for 30 min. The rest of the procedures followed manufacturer’s instruction .The extracted RNA was used within 30 min of extraction or stored at -80°C until use
|
| Label |
cy3
|
| Label protocol |
The Standard Operating Procedures for Aminoallyl Labeling of RNA (ftp://ftp.jcvi.org/pub/data/PFGRC/MAIN/pdf_files/protocols/M007.pdf) from J. Craig Venter Institute was adopted.
|
| |
|
| Channel 2 |
| Source name |
strong biofilm former
|
| Organism |
Listeria monocytogenes |
| Characteristics |
strain background: ScottA
|
| Treatment protocol |
No treatment was applied before extract preparation.
|
| Growth protocol |
Frozen L. monocytogenes cultures were activated by incubating in trypticase soy broth supplemented with 0.5% yeast extract (TSBYE) at 37°C for 18 h, and transferred at least once prior to assessments.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using the RNeasy mini kit (Qiagen, Valencia, CA) following manufacturer’s procedure with slight modification. Briefly, 23.75 ml of fresh TSBYE was inoculated with overnight cultures at 2.5% and grown at 37°C for 6 h until reached at OD600 of 0.6 – 0.7. One mL of aliquots were removed and placed in 4°C refrigerator for 15 min, and the cells were collected by centrifugation at 8000xg for 5 min. The cells were resuspended in 100μL of TE buffer and 6μL of lysozyme (50μg/mL) followed by incubation at 30°C for 30 min. The rest of the procedures followed manufacturer’s instruction .The extracted RNA was used within 30 min of extraction or stored at -80°C until use
|
| Label |
cy5
|
| Label protocol |
The Standard Operating Procedures for Aminoallyl Labeling of RNA (ftp://ftp.jcvi.org/pub/data/PFGRC/MAIN/pdf_files/protocols/M007.pdf) from J. Craig Venter Institute was adopted.
|
| |
|
| |
| Hybridization protocol |
The Standard Operating Procedures for Hybridization of Labeled cDNA probes (ftp://ftp.jcvi.org/pub/data/PFGRC/MAIN/pdf_files/protocols/M008.pdf) by the J. Craig Venter Institute was followed.
|
| Scan protocol |
Microarray slides were scanned on a GenePix 4000B microarray scanner from Molecular Devices. Gain value was acquired in automatic reading mode (3 reads).
|
| Description |
Sample 5
|
| Data processing |
Scanned data from dried slides were normalized and statistical analysis was conducted using the TM4 microarray software suite from J. Craig Venter Institute. Briefly, Midas was used to normalize the microarray using LOWESS normalization. The data were then entered into Multi Experiment Viewer (MEV) where T-Tests were performed using a p-value of p = 0.05. The UID column in MEV file corresponds to to ID column in the gpr file and IA represents Cy3 and IB represents Cy5. Please note that the array which was designed for multiple strains (including Listeria moncytogenes 4b F2365, EGD-e, 1/2a F6854, 4bH7858 and HCC23) was used for this study with the ScottA strain, which resulted in no signal/values for a large percentage of the probes on the array.
|
| |
|
| Submission date |
Sep 04, 2012 |
| Last update date |
Sep 06, 2012 |
| Contact name |
Hua Wang |
| E-mail(s) |
wang.707@osu.edu
|
| Phone |
614-292-0579
|
| Organization name |
The Ohio State University
|
| Department |
Food Science and Technology
|
| Lab |
Hua Wang's lab
|
| Street address |
2015 Fyffe CT
|
| City |
Columbus |
| State/province |
OH |
| ZIP/Postal code |
43210 |
| Country |
USA |
| |
|
| Platform ID |
GPL5854 |
| Series (1) |
| GSE40598 |
Role of a GntR-family response regulator LbrA in Listeria monocytogenes biofilm formation |
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