|
| Status |
Public on Aug 06, 2011 |
| Title |
Spyogenes_M49_ strain 591_CDM_0.4_repl1 |
| Sample type |
RNA |
| |
|
| Source name |
Spyogenes_M49_ strain 591_CDM_0.4
|
| Organism |
Streptococcus pyogenes M49 591 |
| Characteristics |
growth phase: exponential
|
| Treatment protocol |
Harvested by centrifugation
|
| Growth protocol |
Cells cultured in chemical defined medium (CDM), at 37°C under a 5% CO2 20% O2 atmosphere, collected at described OD
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total bacterial RNA from cultures grown to corresponding OD was isolated using the FastRNA ProBlue Kit from MP Biomedicals as outlined in the protocol provided by the manufacturer. The purified total RNA was digested with DNase to remove remaining traces of chromosomal DNA. The RNA preparation was treated with 10 U of DNase for 30 min at 37°C. The enzyme was subsequently heat inactivated at 72°C for 5 min. 5 µg of total RNA were fractionated using the Ambion FlashPAGE Fractionator, Ambion® FlashPAGE Precats Gels, and the Ambion® FlashPAGE™ Buffer Kit, following the manufacturer’s instructions. To collect a fraction of RNA molecules <200 nucleotides, the protocol was slightly modified: the running time was increased from 12 min to up to 45 min at 75 Volt. The fraction of small RNAs was ethanol-precipitated over night at -20°C.
|
| Label |
Cy3
|
| Label protocol |
The RNA was pelleted by centrifugation, dissolved in nuclease-free water, and labelled using the Ambion mirVana labeling Kit (Applied Biosystems, Foster City, CA) following the manufacturer’s instructions.
|
| |
|
| Hybridization protocol |
All hybridization and detection steps were done using the febit’s Geniom RT Analyzer (febit biomed, Heidelberg, Germany). Hybridizations were performed overnight (16 hours) at 42°C. Subsequently, biotin was detected with streptavidin-phycoerythrin (SAPE). A signal amplification step was added using biotinylated anti-streptavidin antibodies (Vector Laboratories, Burlingame, CA, USA) and a second incubation with SAPE (Invitrogen, Carlsbad, CA, USA).
|
| Scan protocol |
Signal detection using the appropriate filter set (570 nm) of the Geniom device employed the autoexposure function of the Geniom software.
|
| Data processing |
Genedata Expressionist software (version 5.3) was used for data processing. Raw data were Quantile normalized applying standard settings (Averaging: Logarithmic Mean) of Expressionist Analyst.
|
| |
|
| Submission date |
Aug 05, 2011 |
| Last update date |
Aug 09, 2011 |
| Contact name |
Nadja Patenge |
| E-mail(s) |
[email protected]
|
| Phone |
493814945916
|
| Fax |
493814945902
|
| Organization name |
University of Rostock
|
| Department |
Institute of Medical Microbiology, Virology and Hygiene
|
| Street address |
Schillingallee 70
|
| City |
Rostock |
| ZIP/Postal code |
18057 |
| Country |
Germany |
| |
|
| Platform ID |
GPL14116 |
| Series (1) |
| GSE31228 |
Identification of small non coding RNAs in S. pyognes M49 using intergenic tiling arrays |
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