|
| Status |
Public on Dec 09, 2008 |
| Title |
GGS27_BR2_2S_12896223annot_MDS_MDS.tav.refIsIA.out |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Geobacter sulfurreducens strain DL1 cell cultures in chemostats under conditions of acetate as the sole electron donor and limiting factor and fumarate as the sole electron acceptor
|
| Organism |
Geobacter sulfurreducens |
| Characteristics |
Strain DL1, grown under steady state conditions in chemostats
Reference
|
| Biomaterial provider |
A Esteve-Nunez, DR Lovley
|
| Treatment protocol |
G. sulfurreducens was anaerobically cultured in chemostats under two conditions: 1) in which acetate was the sole electron donor and the limiting growth substrate, and fumarate was the sole electron acceptor and 2) acetate was the sole electron donor and supplied in excess while fumarate was the sole electron acceptor and the substrate that limited growth.
|
| Growth protocol |
General growth conditions of the G. sulfurreducens DL1 cells were that they were cultured anaerobically in chemostats. Two growth conditions were tested. In the first, the electron donor (acetate 5mM) was limiting
and fumarate was the electron acceptor. In the second condition, acetate as sole electron donor was provided in excess and fumarate was the limiting substrate. A dilution rate of 0.05 per hour was used for all chemostats. Additional details concerning the growth of G. sulfurreducens are given in the following reference: Esteve-Nunez, A., M. Rothermich, M. Sharma, and L. D. 2005. Growth of Geobacter sulfurreducens under nutrient-limiting conditions in continuous culture. Environ. Microbiol. 7(5):641-648.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted as previously described by first mechanically disrupting cells using a FastPrep instrument (Qbiogene, Carlsbad, CA) with Lysing Matrix B (Qbiogene, Carlsbad, CA), followed by nucleic acid
extraction with TRIzol reagent (Invitrogen, Carlsbad, CA) (a monophasic solution of phenol and guanidine isothiocyanate) (Methe et al. 2005). Any residual DNA was removed using RNase-free DNase according to the
manufacturer’s instructions (Ambion, Austin, TX), and treated RNA was subsequently cleaned and concentrated with RNeasy minicolumns (QIAGEN Inc., Valencia, CA). The quality of total RNA was assessed by agarose-formaldehyde gel electrophoresis and the oncentration determined using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE).
|
| Label |
Cy3
|
| Label protocol |
cDNA was labeled from total RNA with Cyanine-3 (Cy-3) and Cyanine-5 (Cy-5) via an indirect coupling method. First cDNA was synthesized from total RNA with aminoallyl-dUTP incorporated into the first strand cDNA to which Cy-3 and Cy-5 fluorescent dye molecules are subsequently coupled.
|
| |
|
| Channel 2 |
| Source name |
Geobacter sulfurreducens strain DL1 cell cultures in chemostats under conditions of acetate was supplied in excess with fumarate supplied as the sole electron acceptor and limiting factor
|
| Organism |
Geobacter sulfurreducens |
| Characteristics |
Strain DL1, grown under steady state conditions in chemostats
Query
|
| Biomaterial provider |
A Esteve-Nunez, DR Lovley
|
| Treatment protocol |
G. sulfurreducens was anaerobically cultured in chemostats under two conditions: 1) in which acetate was the sole electron donor and the limiting growth substrate, and fumarate was the sole electron acceptor and 2) acetate was the sole electron donor and supplied in excess while fumarate was the sole electron acceptor and the substrate that limited growth.
|
| Growth protocol |
General growth conditions of the G. sulfurreducens DL1 cells were that they were cultured anaerobically in chemostats. Two growth conditions were tested. In the first, the electron donor (acetate 5mM) was limiting
and fumarate was the electron acceptor. In the second condition, acetate as sole electron donor was provided in excess and fumarate was the limiting substrate. A dilution rate of 0.05 per hour was used for all chemostats. Additional details concerning the growth of G. sulfurreducens are given in the following reference: Esteve-Nunez, A., M. Rothermich, M. Sharma, and L. D. 2005. Growth of Geobacter sulfurreducens under nutrient-limiting conditions in continuous culture. Environ. Microbiol. 7(5):641-648.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted as previously described by first mechanically disrupting cells using a FastPrep instrument (Qbiogene, Carlsbad, CA) with Lysing Matrix B (Qbiogene, Carlsbad, CA), followed by nucleic acid
extraction with TRIzol reagent (Invitrogen, Carlsbad, CA) (a monophasic solution of phenol and guanidine isothiocyanate) (Methe et al. 2005). Any residual DNA was removed using RNase-free DNase according to the
manufacturer’s instructions (Ambion, Austin, TX), and treated RNA was subsequently cleaned and concentrated with RNeasy minicolumns (QIAGEN Inc., Valencia, CA). The quality of total RNA was assessed by agarose-formaldehyde gel electrophoresis and the oncentration determined using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE).
|
| Label |
Cy5
|
| Label protocol |
cDNA was labeled from total RNA with Cyanine-3 (Cy-3) and Cyanine-5 (Cy-5) via an indirect coupling method. First cDNA was synthesized from total RNA with aminoallyl-dUTP incorporated into the first strand cDNA to which Cy-3 and Cy-5 fluorescent dye molecules are subsequently coupled.
|
| |
|
| |
| Hybridization protocol |
Prehybridization:
1. Prepare prehybridization buffer containing 5x SSC, 0.1% SDS and 1% bovine serum albumin. Place slides to be analyzed into a Coplin jar fill with prehybridization buffer, and incubate at 42 degree Celsius for 45 minutes.
2. Wash slides in distilled water 4-8 times to remove excess prehybridization buffer.
3. Dip the slides in room temperature isoproponol 10 times and dry in slide spinner.
4. Slides should be used immediately following prehybridization. We have found that hybridization efficiency decreases rapidly if the slides are allowed to dry for more than one hour.
Hybridization:
1. Resuspend the probe in 70ul hybridization buffer (50% formamide, 5x SSC, 1mg/ml salmon sperm DNA, syringe filtered).
2. Incubate at 98 degree Celsius for 15 minutes to resuspend as much probe as possible and spin for 5 minutes at maximum angular velocity to collect any remaining solid at the bottom of the tube.
3. Place a polyethylene hydrophobic lifterslip on each prehybridized slide so that it covers the array.
4. Apply the probe to one end of the coverslip and allow capillary action to draw the liquid through to cover the whole array. Place the slide in a sealed hybridization chamber, add 20 ul of 20x SSC to the chamber at each end of the slide.
5. Place the sealed chamber in a 42 degree Celsius water bath and incubate for a minimum 18-20 hours.
6. Remove the array from the hybridization chamber, taking care not to disturb the coverslip.
7. Place the slide in a staining dish with low-stringency wash buffer containing 1x SSC and 0.2% SDS at 42 degree Celsius.
8. Gently remove the coverslip while the slide is in solution and agitate for 2-4 minutes. Wash again in 1xSSC and 0.2% SDS for 2-4 minutes.
9. Wash the slide twice at high-stringency in a staining dish containing 0.1x SSC and 0.2% SDS at room temperature, agitating for 2-4 minutes.
10. Wash the slide twice in 0.1x SSC, agitating for 2-4 minutes.
11. Provide one final wash by dipping each slide 6-7 times in a 0.05x SSC solution immediately prior to drying.
12. Dry the slide in a slide spinner.
|
| Scan protocol |
Slides were promptly scanned post-hybridization at a 10 um resolution using an Axon 4000B scanner with GenePix 4.0 software. PMT values were optimized during scanning to balance channel intensities.
|
| Description |
Information covered in other sections
|
| Data processing |
Processing of 16-bit TIFF images from hybridized arrays were produced using the TIGR TM4 package (www.tm4.org). Intensity values for Cy3 and Cy5 channels were obtained using TIGR-Spotfinder software.
Normalization was performed using the LOWESS algorithm available in TIGR-MIDAS using block mode and a smooth parameter of 0.33. All intensity values less than two times greater than background were removed from subsequent analysis and replicate reporter intensities on one slide (one technical replicate) were reduced to a single value by computing the geometric mean.
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| |
|
| Submission date |
Dec 10, 2007 |
| Last update date |
Aug 14, 2011 |
| Contact name |
Chun-Hua Wan |
| E-mail(s) |
[email protected]
|
| Phone |
301-795-7707
|
| Organization name |
The J. Craig Venter Institute
|
| Department |
Pathogen Functional Genomics Resources Center (PFGRC)
|
| Lab |
IFX
|
| Street address |
9704 Medical Center Dr
|
| City |
Rockville |
| State/province |
MD |
| ZIP/Postal code |
20850 |
| Country |
USA |
| |
|
| Platform ID |
GPL5768 |
| Series (1) |
| GSE9830 |
G. sulfurreducens growth with acetate as limiting electron donor versus fumarate as limiting electron acceptor |
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