|
| Status |
Public on Mar 05, 2013 |
| Title |
M. sedula - Autotrophic Carbon Rich (ACR) vs Heterotrophic (HTR) |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Msed_ACR
|
| Organism |
Metallosphaera sedula DSM 5348 |
| Characteristics |
cell type: M. sedula DSMZ 5348 - whole cells
|
| Growth protocol |
Cells was grown aerobically at 70°C in a shaking oil bath (90 rpm) under autotrophic conditions on DSMZ medium 88 at pH 2 with a headspace concentration of 33% H2, 8% CO2, 12% O2, balance N2. Cell growth was scaled up from 300 ml in sealed 1L bottles to 2 liters in a stirred bench-top glass fermentor (Applikon), also on DSMZ medium 88 (pH 2) at 70°C, with agitation at 250 rpm. Two separately regulated gas feeds were used such that flow rates were held constant for all conditions at 1 ml/min for the hydrogen/CO2 gas mixes (composition varied) and 100 ml/min for air (composition - 78% N2, 21% O2, 0.03% CO2). For the autotrophic carbon rich (ACR) condition the feed was H2 (80%) and CO2 (20%).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
At harvest, cells were quickly chilled and then centrifuged at 6000 x g for 15 minutes at 4C. RNA was extracted using Trizol (Invitrogen) and purified using an RNeasy Mini Kit (Qiagen).
|
| Label |
Cy3
|
| Label protocol |
Purified RNA was reverse transcribed using Superscript III (Invitrogen), re-purified, labeled with Cy3 dye (GE Healthcare), and hybridized to an Mse microarray slide (Corning).
|
| |
|
| Channel 2 |
| Source name |
Msed_HTR
|
| Organism |
Metallosphaera sedula DSM 5348 |
| Characteristics |
cell type: M. sedula DSMZ 5348 - whole cells
|
| Growth protocol |
Cells was grown aerobically at 70°C in a shaking oil bath (90 rpm) under heterotrophic conditions on DSMZ medium 88 supplemented with 0.1% tryptone at pH 2. Cell growth was scaled up from 300 ml in 1L bottles to 2 liters in a stirred bench-top glass fermentor (Applikon), also on DSMZ medium 88 with 0.1% tryptone (pH 2) at 70°C, with agitation at 250 rpm. Two separately regulated gas feeds were used such that flow rates were held constant for all conditions at 1 ml/min for the hydrogen/CO2 gas mixes (composition varied) and 100 ml/min for air (composition - 78% N2, 21% O2, 0.03% CO2). For the heterotrophic (HTR) condition the feed was N2 (80%) and CO2 (20%).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
At harvest, cells were quickly chilled and then centrifuged at 6000 x g for 15 minutes at 4C. RNA was extracted using Trizol (Invitrogen) and purified using an RNeasy Mini Kit (Qiagen).
|
| Label |
Cy5
|
| Label protocol |
Purified RNA was reverse transcribed using Superscript III (Invitrogen), re-purified, labeled with Cy5 dye (GE Healthcare), and hybridized to an Mse microarray slide (Corning).
|
| |
|
| |
| Hybridization protocol |
Sample preparation steps for microarray hybridization: Prepare Prehyb buffer: 225mL water, 75mL 20X SSC, 3ml 10% SDS, 0.3g BSA. Preheat Prehyb buffer 45 min 42 degree C. Prehybidize slides at 42 degree C for 45 min. Wash slides by dipping 5 time in sterile dH2O at RT. Dip twice in isopropanol at RT. Dry slide in slide spinner. Add 1 ul COT1-DNA (20mg/ml) to samples. Heat probe mixture to 95C in heat block for 2 min to denature DNA, do a quick spin. Add 30 ul of 2x hyb. buffer that has been preheated to 42C to each tube. Hybridization buffer: 27.2uL water, 20uL 20X SSC, 8uL 50X Denhardts solution, 24uL formamide, 0.8uL 10% SDS. Apply hyb. mix to slide and cover with a 20x60 mm polyethylene hydrophobic coverslip. Place slide in water proof container, cover well with foil and place in 42C bath for 18-20 hours. The following is a standard hybridization method that seems to work well for this procedure: 1 wash cycle at 42C with 2X SSC + 0.2% SDS; 1 wash cycle at RT with 0.5X SSC + 0.2% SDS; 1 wash cycles at RT with 0.5X SSC; final wash with 0.05X SCC.
|
| Scan protocol |
Images were aquired with a GenePix 4000B with automatic calculation of PMT gain.
|
| Description |
Scatterplots of signal intensities are reviewed to check for balance between channels.
|
| Data processing |
Signals for each channel were obtained using GenePix Pro v6.0 software using histogram quantitation and total normalization options to produce raw data, followed by manual verification of spot identification. Once all slides are quantitated, data from all slides in loop were analyzed with JMP Genomics 3.1 (SAS, NC), using a mixed effects ANOVA model [Wolfinger et al. J Comput Biol 8 625-637 (2001).
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| |
|
| Submission date |
Aug 07, 2012 |
| Last update date |
Mar 05, 2013 |
| Contact name |
Aaron Hawkins |
| E-mail(s) |
abhawki2@ncsu.edu
|
| Phone |
9195154452
|
| Organization name |
North Carolina State Universit
|
| Department |
Chemical and Biomolecular Engineering
|
| Lab |
Kelly Group
|
| Street address |
840 Main Campus Dr
|
| City |
Raleigh |
| State/province |
North Carolina |
| ZIP/Postal code |
27606 |
| Country |
USA |
| |
|
| Platform ID |
GPL6785 |
| Series (1) |
| GSE39944 |
M. sedula transcriptional response under "strict" carbon-limited autotrophy compared to carbon "rich" autotrophy and heterotrophy |
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