|
| Status |
Public on Aug 01, 2013 |
| Title |
1H -N Kb3 |
| Sample type |
RNA |
| |
|
| Source name |
Karenia brevis, N- limited, newly transcribed RNA during 1 h
|
| Organism |
Karenia brevis |
| Characteristics |
isolate: Wilson Isolate
[no3] added on day 9: 0 µM
4-thiouracil labeling time: 1 h
|
| Treatment protocol |
155 µM NO3 was added to stationary phase (Day 9) cultures grown in 10 µM NO3 (n=6). 0.2 mM 4-thiouracil in DMSO was added for 1 h during the first (n=3) or fourth (n=3) hour post-N-addition to cultures to biosynthetically label newly synthesized RNA. Additionally, 0.2 mM 4-thiouracil in DMSO was added for 1 h to stationary phase (Day 9) cultures grown in 10 µM NO3 (n=3).
|
| Growth protocol |
Karenia brevis, Wilson isolate, was grown in 1 L batch cultures in autoclaved, 20 µm filtered seawater at 36 ‰ enriched with f/2 medium, modified to contain only 10 µM NO3, at 25 °C on a 16 h:8 h light:dark cycle with illumination from cool white lights at 150 µE m-2 sec-1
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cultures were independently harvested by centrifugation. Total RNA from each sample was prepared using TriReagent. Following precipitation in ethanol, the RNA was resuspended and purified using a Qiagen RNeasy column. Total RNA was quantified using a NanoDrop ND-1000 and qualified on an Agilent 2100. Total RNA was biotinylated with Pierce EZ-link Biotin-HPDP, precipitated in ethanol, and quantified using a NanoDrop ND-1000. This RNA was then separated into pre-exisiting (non-biotinylated) and newly synthesized (biotinylated) RNA pools using streptavidin coated magnetic beads. Once separated, the newly synthesized RNA fraction was precipitated with isopropanol and quantified using a NanoDrop ND-1000 and qualified on an Agilent 2100.
|
| Label |
Cy3
|
| Label protocol |
Newly synthesized RNA (25 ng) was amplified and labeled with Cy3 dye using the Low Input Quick Amp Labeling Kit (Agilent, Santa Clara, CA). The amplified, labeled RNA was quantified using a NanoDrop ND-1000.
|
| |
|
| Hybridization protocol |
480 ng of Cy3 labeled targets were hybridized to the array for 17 hours at 60°C. After hybridization, arrays were washed according to the manufacturer’s protocol (Agilent, Santa Clara, CA).
|
| Scan protocol |
Microarrays were imaged using an Agilent microarray scanner using extended dynamic range scanning
|
| Data processing |
Images were extracted with Agilent Feature Extraction version 9.5.3.1 using a rank consistency filter and a combination linear and LOWESS normalization algorithm.
|
| |
|
| Submission date |
Apr 18, 2013 |
| Last update date |
Aug 01, 2013 |
| Contact name |
Frances M Van Dolah |
| E-mail(s) |
[email protected]
|
| Organization name |
Marine Biotoxins Program, NOAA Center for Coastal Environmental Health and Biomolecular Research
|
| Street address |
219 Fort Johnson Rd
|
| City |
Charleston |
| State/province |
SC |
| ZIP/Postal code |
29412 |
| Country |
USA |
| |
|
| Platform ID |
GPL13364 |
| Series (1) |
| GSE46175 |
Global Analysis of de novo Transcription Following Biosynthetic Labeling in a Dinoflagellate, Karenia brevis |
|
