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| Status |
Public on May 29, 2013 |
| Title |
Infected cells from Medicago truncatula root nodules replicate 3 |
| Sample type |
RNA |
| |
|
| Source name |
Infected cells from Medicago truncatula root nodules
|
| Organism |
Medicago truncatula |
| Characteristics |
tissue: Root nodule
genotype: Jemalong A17
age: 21 days
|
| Treatment protocol |
Exised nodules were fixed in Farmer's fixative(3:1 ethanol:acetic acid),incubated at 4 0C overnigh tafter 30 min. vacuum. Fixed nodules were further dehydrated through an ethanol series: 75%, 85%, 100% (4x) for 15 min. each at room temperature (RT). At the first 100% ethanol step eosin B was added to facilitate the recognition of the nodule meristem during the sectioning steps. Nodules were subsequently infiltrated with xylene: ethanol 1:3, 1:1, 3:1 and finally 100% xylene (3x); 30 min at RT each. Next, the nodules were infiltrated with liquid filtered paraffin (Paraplast) at 60 0C for 2 days including 4 changes of paraffin. After solidification, 8 µm sections were cut using a microtome. Section were deparaffinized using 100% xylene 2x 5 min. each, air dryed and immediately used for laser capture using a PixCell II LCM system (Arcturus).
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| Growth protocol |
Medicago truncatula A17 plants were grown in (agra)perlite saturated with nitrate-free Fähraeus medium and inoculated with 2 ml a suspension (OD600 0.1) of Sinorhizobium meliloti strain Sm2011 per plant. Nodules were harvested 3 weeks after inoculation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Captured cells (3 biological replicates for each cell-/tissue type) were stored in 350 µl RLT buffer (containing β-mercaptoethanol and 50 ng poly-Inosine ) from the Qiagen RNeasy Micro kit and stored at - 80 0C until RNA extracion. RNA was extracted using the Qiagen Qiagen Rneasy Micro kit according to the manufacturers instructions, including an on column DNAse treatment.
|
| Label |
biotin
|
| Label protocol |
RNA samples were amplified according to the first amplification cycle of the Affymetrix Two-cycle Target Labeling kit user manual. Briefly, total RNA containing spiked-in poly-A+ RNA controls was used in a reverse transcription reaction (Two-cycle Target Labeling kit; Affymetrix, Santa Clara, CA, USA) to generate first-strand cDNA. After second-strand synthesis, double-stranded cDNA was used in a 16 h in vitro transcription (IVT) reaction to generate aRNA (Two-cycle Target Labeling kit). The generated aRNA samples were than processed according to the Affymetrix GeneChip 3’ IVT Express kit user manual. Briefly, 100 ng of aRNA was used in a reverse transcription reaction (GeneChip 3’ IVT Express Kit; Affymetrix, Santa Clara, CA, USA) to generate first-strand cDNA. Double-stranded cDNA obtained by second-strand synthesis was then used in a 16 h IVT reaction to generate aRNA (GeneChip 3’ IVT Express Kit). Size distribution of in vitro transcribed aRNA and fragmented aRNA, respectively, was assessed via an Agilent 2100 Bioanalyzer (Agilent, Böblingen, Germany), using an RNA 6000 Nano Assay. Assay.
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| |
|
| Hybridization protocol |
30-40 μg of fragmented aRNA was added to a 250 μl hybridization cocktail also containing hybridization controls. 200 μl of the mixture was hybridized on GeneChips for 16 h at 45°C. Standard post hybridization wash and double-stain protocols (FS450_0001; GeneChip HWS kit; Affymetrix, Santa Clara, CA, USA) were used on an Affymetrix GeneChip Fluidics Station 450.
|
| Scan protocol |
GeneChips were scanned on an Affymetrix GeneChip scanner 3000 7G.
|
| Description |
Mt_MtGeneChip0185_A143_jbecker_IGC_PP _(Medicago)
Gene expression data from infected cells from Medicago truncatula root nodules
|
| Data processing |
Cel files obtained from Medicago GeneChip hybridizations were analysed using packages from the Bioconductor project. Normalization was performed across all GeneChips using the Robust Multiarray Average (RMA) algorithm using the empirical Bayes approach to adjust for background in the Bioconductor library “gcrma.”. Only the 5 most 3’located probe sets on the GeneChip were used to account for observed 3’bias.
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| |
|
| Submission date |
Jan 08, 2013 |
| Last update date |
May 29, 2013 |
| Contact name |
Helge Küster |
| E-mail(s) |
[email protected]
|
| Organization name |
Leibniz Universität Hannover
|
| Department |
Institut für Pflanzengenetik
|
| Lab |
Abt. IV - Pflanzengenomforschung
|
| Street address |
Herrenhäuser Str. 2
|
| City |
Hannover |
| ZIP/Postal code |
30419 |
| Country |
Germany |
| |
|
| Platform ID |
GPL4652 |
| Series (1) |
| GSE43354 |
Cell- and tissue-specific transcriptome analyses of Medicago truncatula root nodules |
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