|
| Status |
Public on Nov 17, 2009 |
| Title |
Bacteroids three weeks |
| Sample type |
RNA |
| |
|
| Source name |
Wildtype R. etli CFN42 bacteroids harvested 3 weeks after inoculation
|
| Organism |
Rhizobium etli CFN 42 |
| Characteristics |
genetic background: CFN 42
genotype: wild type
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using the TRIzol Plus RNA Purification kit (Invitrogen). Bacteroid pellets were resuspended in 1 ml of TRIzol and shaken twice by a Precellys 24 (Bertin Technologies) at 6500 rpm for 45 seconds with 0.25 ml of 0.1 mm glass beads before following the manufacturer’s instructions. Phase Lock Gel tubes (heavy type) were used to efficiently separate the organic and aqueous phase. DNA contamination was removed by two treatments of 2 μl TURBO DNase (Ambion) and afterwards checked by PCR (45 cycles). ). To increase RNA yields and account for experimental variation, RNA from 6 different free-living cultures was pooled. Bacteroid RNA was obtained after crushing 10g nodules of 25 plants using liquid nitrogen. RNA was precipitated in 3 volumes of isopropanol and 1/10 volume of sodium acetate, washed twice in ethanol and dissolved in nuclease-free ultrapure water. RNA integrity was analyzed using Experion RNA StdSens Chips (Biorad, Hercules, CA, before and after precipitation ). The sample had an RNA Quality Indicator value of 10. The ncRNA peak could be detected in each sample. RNA quantity and purity was assessed using the NanoDrop ND-1000. The A260/A280 ratio and A260/A230 ratio was ≥ 2.
|
| Label |
Cy3
|
| Label protocol |
cDNA, median≥400bp, was synthesized using random decamers (Ambion) and the SuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen) according to the manufacturer’s protocol. All cDNA samples were labeled with Cy3 by Nimblegen using the NimbleGen One- Color DNA Labeling Kit (05223555001).
|
| |
|
| Hybridization protocol |
The Cy3-labeled cDNA was hybridized to the custom R. etli tiling expression array at 42°C for 16 hour using the NimbleGen Hybridization System and kit.
|
| Scan protocol |
GenePix 4000B.
|
| Description |
R. etli CFN42 was grown at 30˚C in AMS medium supplied with 10 mM NH4Cl and 10 mM succinate while monitoring the optical density (OD) of the culture. Bacteroid samples were obtained from nodules 3 weeks after inoculation of Common bean plants (Phaseolus vulgaris cv Limburgse vroege).
|
| Data processing |
Data preprocessing was done by performing a nonlinear intensity dependent rescaling on non-background corrected data. To this end, a loess fit normalization with a span of 25% was performed for each array compared against an artificial reference array, consisting of the median intensity values of each probe across all arrays. To ensure that the artificial reference itself was not altered by this rescaling, the artificial reference expression levels were chosen for the average log intensity in the loess fit (instead of the mean expression levels of the respective array and the artificial reference). A robust median-polish procedure was used to combine measurements from multiple probes into a single value. Results are linked as supplementary GFF files in standard format with columns: seqname (chromosome name); source (article); feature (RNA); start; end; score (normalized value); strand (forward (1) or reverse (-1)); frame (empty for RNA); attribute (ID).
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| |
|
| Submission date |
Oct 15, 2009 |
| Last update date |
Nov 03, 2010 |
| Contact name |
Kristof Engelen |
| Organization name |
KULeuven
|
| Lab |
CMPG
|
| Street address |
Kasteelpark Arenberg 20
|
| City |
Leuven |
| ZIP/Postal code |
3001 |
| Country |
Belgium |
| |
|
| Platform ID |
GPL9409 |
| Series (2) |
| GSE18580 |
sRNAs in Rhizobium etli differentially expressed during free-living and host-associated growth |
| GSE25094 |
Transcriptome analysis of Rhizobium etli’s free-living and symbiotic non-growing state. |
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