|
| Status |
Public on Aug 01, 2013 |
| Title |
CC9311_EB-shock_replicate 3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Synechococcus sp. CC9311, no treatment
|
| Organism |
Synechococcus sp. CC9311 |
| Characteristics |
strain: CC9311
treatment: no treatment
|
| Treatment protocol |
Duplicate 1.5-liter batch cultures of WH8102 and CC9311 were grown up to the early exponential phase and half the culture was spun down at room temperature, resuspended in Trizol reagent (Invitrogen), and frozen at -80°C for RNA extraction in parallel with treated cells, taking care that the harvesting time stayed under 30 min. EB (final concentration 2 μg/mL) or MC (final concentration 0.5 μg/mL) were added to the remaining culture. After a two hour incubation, these cells were harvested and suspended in Trizol reagent as above.
|
| Growth protocol |
Axenic Synechococcus sp. WH8102 and Synechococcus sp. CC9311 cultures were maintained in either SN medium or in an artificial seawater medium (SOW) with 9.0 mM NaNO3 standard. For growth assays, batch cultures were grown in glass flasks, gently stirred, or in glass tubes, at 25 °C under 30 μEinstein m-2 s-1 continuous white light.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Trizol reagent (Invitrogen) according to the manufacturer’s instructions, RNA was resuspended in 100 μl of DEPC-treated water, further processed using RNeasy Mini kit (Qiagen) following the manufacturer’s protocol, twice digested with DNase I (Qiagen), then eluted from the columns.
|
| Label |
Cy3
|
| Label protocol |
Reverse transcription of RNA samples was performed using the SuperScript Plus Indirect cDNA Labeling System (Invitrogen) with random hexamer primers. Approximately 4 µg of total RNA was used for indirect labelling, leading to the production of approximately 4 µg of cDNA with approximately 200 pmol of dye molecule incorporated per microgram of cDNA synthesized.
|
| |
|
| Channel 2 |
| Source name |
Synechococcus sp. CC9311, EB treated
|
| Organism |
Synechococcus sp. CC9311 |
| Characteristics |
strain: CC9311
treatment: EB treated
|
| Treatment protocol |
Duplicate 1.5-liter batch cultures of WH8102 and CC9311 were grown up to the early exponential phase and half the culture was spun down at room temperature, resuspended in Trizol reagent (Invitrogen), and frozen at -80°C for RNA extraction in parallel with treated cells, taking care that the harvesting time stayed under 30 min. EB (final concentration 2 μg/mL) or MC (final concentration 0.5 μg/mL) were added to the remaining culture. After a two hour incubation, these cells were harvested and suspended in Trizol reagent as above.
|
| Growth protocol |
Axenic Synechococcus sp. WH8102 and Synechococcus sp. CC9311 cultures were maintained in either SN medium or in an artificial seawater medium (SOW) with 9.0 mM NaNO3 standard. For growth assays, batch cultures were grown in glass flasks, gently stirred, or in glass tubes, at 25 °C under 30 μEinstein m-2 s-1 continuous white light.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Trizol reagent (Invitrogen) according to the manufacturer’s instructions, RNA was resuspended in 100 μl of DEPC-treated water, further processed using RNeasy Mini kit (Qiagen) following the manufacturer’s protocol, twice digested with DNase I (Qiagen), then eluted from the columns.
|
| Label |
Cy5
|
| Label protocol |
Reverse transcription of RNA samples was performed using the SuperScript Plus Indirect cDNA Labeling System (Invitrogen) with random hexamer primers. Approximately 4 µg of total RNA was used for indirect labelling, leading to the production of approximately 4 µg of cDNA with approximately 200 pmol of dye molecule incorporated per microgram of cDNA synthesized.
|
| |
|
| |
| Hybridization protocol |
Microarray slides were immersed in pre-hybridization buffer (1 % BSA, 5X SSC, 0.1 % SDS, 0.45 mm-filtered) before hybridization. Slides were washed with sterile water (4x) and isopropanol (3x), spin-dried and covered with lifterslip. cDNA probes were dried using Speedvac and then resuspended in hybridization buffer (50 % formamide, 5X SSC, 0.1 % SDS, 0.45 mm-filtered). Probes were heated at 95 °C (15 min) before applying to the slides. Hybridization was performed overnight at 42°C.
|
| Scan protocol |
Slides were scanned at 10-um resolution using an Axon 4000B scanner with GenePix 4.0 software
|
| Data processing |
Processing of the TIFF images from hybridized arrays was performed using TIGR-Spotfinder (www.tigr.org/software), and the datasets normalized by applying the LOWESS algorithm, using block mode and a smooth parameter of 0.33, available in the TIGR-MIDAS package (www.tigr.org/software). Statistical analysis was performed on the mean of log2-transformed signal ratios of the replicate spots using the Statistical Analysis of Microarrays (SAM) algorithms with a false discovery rate of less than 1%.
|
| |
|
| Submission date |
Aug 01, 2012 |
| Last update date |
Aug 01, 2013 |
| Contact name |
Sasha Tetu |
| Organization name |
Macquarie University
|
| Department |
CMBS
|
| Street address |
North Ryde
|
| City |
Sydney |
| State/province |
NSW |
| ZIP/Postal code |
2019 |
| Country |
Australia |
| |
|
| Platform ID |
GPL15878 |
| Series (1) |
| GSE39818 |
Open Ocean and Coastal Strains of Marine Synechococcus display distinctly different Global Responses to DNA Damaging Agents |
|
