|
| Status |
Public on Nov 01, 2013 |
| Title |
HeterotrophicCulture_WholeFilament_rep3 |
| Sample type |
RNA |
| |
|
| Source name |
HeterotrophicCulture_WholeFilament
|
| Organism |
Trichormus variabilis ATCC 29413 |
| Characteristics |
genotype: Wild-type
phenotype: whole filament
accession: NC_007410 + NC_007411 + NC_007412 + NC_007413
|
| Growth protocol |
Anabaena variabilis ATCC 29413 was grown in an eightfold dilution of the medium of Allen and Arnon (Allen and Arnon, Plant Physiol. 30:366) on a shaker at 30°C and 140 rpm. Phototrophic and mixotrophic cultures were grown under continuous illumination by cool white fluorescent lamps (60−70 µmol photons m-2 s-1). Mixotrophic cultures were supplemented with 5 mM fructose. Heterotrophic cultures were grown in the dark in the presence of 5 mM fructose. Cultures were harvested after seven days for phototrophic and heterotrophic cultures, and after four days for mixotrophic cultures
|
| Extracted molecule |
total RNA |
| Extraction protocol |
separation protocol: Cells pellets from 400-mL cultures were resuspended in 15 mL RNAlater solution (Ambion) and stored at -80°C prior to use. Thawed filament suspensions were sedimented, resuspended in 50 mL of N2-sparged HP buffer (30 mM Hepes/30 mM Pipes/1 mM MgCl2, pH 7.2), and washed three times with HP buffer containing 10 mM Na2EDTA (HP/EDTA). 20% of the filament suspension was used to extract RNA from whole filaments. The rest was used to separate vegetative cell-specific extracts and heterocysts. The washed filaments were resuspended in 40 mL HP/EDTA containing 1 mg mL-1 lysozyme and were shaken at 30°C for 5 min. The lysozyme-treated suspension was sedimented and resuspended in 10 mL HP buffer. The suspension, in a test tube, was subjected to cavitation (Ultrasonic Cleaner bath, Cole-Palmer) for 3 min to destroy a fraction of the vegetative cells. Heterocysts and remaining vegetative cells were sedimented and the supernatant (vegetative cell lysate) was saved on ice for extraction of vegetative cell-specific RNA. The sedimented cells were washed twice with HP/EDTA. The washed cells were resuspended in 1 mL HP/EDTA containing 0.2 mg mL-1 lysozyme and were shaken at 30°C for 25 min. This lysozyme-treated suspension was sedimented and resuspended with 1 mL HP. This suspension was immersed in a 12°C sonic bath for 15 min to destroy all remaining vegetative cells. After centrifugation, the supernatant solution was discarded, the heterocyst-containing pellet was washed three times with HP and used as the source of heterocyst-specific RNA.
Total RNA was extracted from whole filaments, purified heterocysts, and vegetative cell extracts with the RiboPure-Bacteria kit (Ambion). Extracted RNA was purified with an RNeasy Mini kit (Qiagen) and eluted in 30 µL water. RNA concentration was determined on a NanoDrop ND-1000 spectrophotometer. RNA quality was determined by analysis with an Agilent 2100 bioanalyzer.
|
| Label |
Cy3
|
| Label protocol |
Labeling was performed by NimbleGen Systems Inc., Madison, WI USA, following their standard operating protocol. See www.nimblegen.com.
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| |
|
| Hybridization protocol |
Hybridization was performed by NimbleGen Systems Inc., Madison, WI, USA following their standard operating protocol. See www.nimblegen.com.
|
| Scan protocol |
Scanning was performed by NimbleGen Systems Inc., Madison, WI USA, following their standard operating protocol. See www.nimblegen.com.
|
| Description |
This sample is of whole filaments of Anabaena variabilis ATCC 29413 in heterotrophic culture. It is the third of three wild-type biological replicates used in this experiment, each from separate cultures.
|
| Data processing |
The raw data (.pair file) was subjected to RMA (Robust Multi-Array Analysis; Irizarry et al. Biostatistics 4(2):249), quantile normalization (Bolstad et al. Bioinformatics 19(2):185), and background correction as implemented in the NimbleScan software package, version 2.4.27 (Roche NimbleGen, Inc.).
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| |
|
| Submission date |
Apr 15, 2013 |
| Last update date |
Nov 01, 2013 |
| Contact name |
Claire Vieille |
| E-mail(s) |
[email protected]
|
| Phone |
517-884-5392
|
| Organization name |
Michigan State University
|
| Department |
Microbiology and Molecular Genetics
|
| Street address |
567 Wilson Rd
|
| City |
East Lansing |
| State/province |
MI |
| ZIP/Postal code |
48824 |
| Country |
USA |
| |
|
| Platform ID |
GPL15883 |
| Series (1) |
| GSE46076 |
Cell-specific gene expression in Anabaena variabilis grown phototrophically, mixotrophically, and heterotrophically |
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