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| Status |
Public on Oct 31, 2017 |
| Title |
UV_1 |
| Sample type |
RNA |
| |
|
| Source name |
Polynucleobacter asymbioticus strain QLW-P1DMWA-1T
|
| Organism |
Polynucleobacter asymbioticus |
| Characteristics |
strain: QLW-P1DMWA-1T
|
| Treatment protocol |
Once the bacterial samples reached OD = 0.1, they were subjected to three experimental scenarios. One batch continued rotating at 26ºC. Second batch was transferred to 4ºC incubator, set at 200 rpm. Third batch (already growing at 26ºC) was periodically treated with 30 min. of UV irradiation (two times a day). Entire experiment was run for three days. Cells were harvested after 72 hrs. (OD ~ 0.25) by treating with RNAlaterTM stabilization solution. Cell pellets were transferred to -80ºC freezer till whole RNA extraction.
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| Growth protocol |
Polynucleobacter asymbioticus strain QLW-P1DMWA-1T was cultured in nutritionally rich liquid R2A media. Incubation was performed at 26ºC, in UV permeable polyethylene bags (~35% permeability). Rotation of the incubator was set at 200 rpm. Bacteria was allowed to grow until they reached an O.D. (260nm) of 0.1. Afterwards, they were subjected to three experimental treatments. Three treatments were (a). 26ºC, (b). 26ºC with UV irradiation, and (c). 4ºC incubation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Whole RNA extraction was performed with the NucleoSpin® RNA kit (Macherey-Nagel GmbH & Co. KG; Düren, Germany) according to the RNA isolation protocol provided by the company.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.2 ug RNA using the One-Color Quick Amp Labeling (Agilent) and the Full Spectrum Multistart Primer (BioCat GmbH, Heidelberg, Germany) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Hilden, Germany). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
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| |
|
| Hybridization protocol |
0.625 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 µl containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 25 µl of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Agilent P. necessarius 8x15K Microarrays (G48102) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by using Acetonitrile.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2565CA) using one color scan setting for 8x15K array slides (Scan Area 61x21.6 mm, Scan resolution 3um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software (version 10.5.1.1) (Agilent) using default parameters (protocol GE1_107_Sep09 and Grid: 048102_D_F_20130312) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
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| |
|
| Submission date |
Apr 24, 2017 |
| Last update date |
Jan 23, 2018 |
| Contact name |
Abhishek Srivastava |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Vienna
|
| Street address |
Althanstrasse 14
|
| City |
Vienna |
| ZIP/Postal code |
1090 |
| Country |
Austria |
| |
|
| Platform ID |
GPL23363 |
| Series (1) |
| GSE98129 |
Transcriptional analysis of freshwater bacterium Polynucleobacter asymbioticus strain QLW-P1DMWA-1T |
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