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| Status |
Public on Feb 06, 2015 |
| Title |
Mycorrhizal protocorm-3 |
| Sample type |
SRA |
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| Source name |
Mycorrhizal protocorm
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| Organism |
Tulasnella calospora |
| Characteristics |
host: Serapias vomeracea
tissue: Mycorrhizal protocorm
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| Growth protocol |
The fungal isolate is T. calospora strain AL13, deposited at the Mycotheca Universitatis Taurinensis collection (MUT4182) at the Department of Life Sciences and Systems Biology, University of Turin, Italy. This strain was isolated from the roots of Anacamptis laxiflora collected in a meadow in northern Italy (Festuco-Brometalia, at 410–450 m a.s.l.). The fungus was grown as a free living culture for 14 days at 24°C on Oatmeal-agar medium (3% oat flakes, 1.5% agar); ten plugs of 0.5 cm of diameter were then aseptically collected from the actively growing margin of the colony, and inoculated in 100mL Erlenmeyer flasks containing 50 mL of liquid Oatmeal modified medium (0.75% oat flakes). Flasks were then incubated on an orbital shaker at 150 rpm for 14 days at 24°C, and three separate RNA extractions were performed. Symbiotic germination of Serapias vomeracea seeds was performed in plastic petri plates (9 cm in diameter, 1.5 cm in height) containing Oatmeal-agar medium (0.3% milled oat, 2.0% agar) inoculated with T. calospora. Protocorms were incubated at 20°C in darkness and collected 30 days after inoculation, immediately frozen in liquid nitrogen and stored at -80°C before RNA extraction.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from 200 mg aliquots of T. calospora free living mycelia grown in Oatmeal modified liquid medium. Mycelium was mechanically ground in liquid nitrogen and RNA was extracted in Tris-HCl extraction buffer (100 mM Tris-HCl pH 8, 100 mM NaCl, 20 mM Na-EDTA, 0.1% PVP, 1% Na-laurylsarcosine sodium salt dissolved in DEPC-treated deionised water), mixed 1:1 with phenol (pH 4.5-5; Roti- Phenol, Roth A980). A phenol: chloroform: Isoamyl alcohol (25:24:1, v/v/v) and a chloroform extraction followed, with 5 min centrifugation at 14000 rpm and 4°C after each extraction step. RNA was precipitated with isopropyl alcohol at -80°C for 30 min, followed by 30 min centrifugation at 14000 rpm and 4°C. The pellet was then resuspended in DEPC treated water: 6M LiCl solution (1:1, v/v) and precipitated overnight at 4°C. After 30 min centrifugation at 14000 rpm and 4°C, the RNA was rinsed with 70% ethanol, centrifuged for 5 min at 14000 rpm and 4°C, air dried on ice, re-suspended in DEPC-treated water and quantified using Nanodrop 1000 (Thermo Scientifics, USA) and Qubit 2.0 (Life Technologies, Italy). RNA integrity was checked using a Bioanalyzer 2100 (Agilent Technologies, Italy). Total RNA was extracted from mycorrhizal S. vomeracea protocorms using the CTAB method. 100 mg aliquots of well-developed protocorms were mechanically ground in liquid nitrogen and RNA was extracted in CTAB buffer (2% CTAB, 2% PVP, 100 mM Tris-HCl pH 8.0; 25 mM EDTA pH 8; 2 M NaCl) at 65°C. 2% PVPP was added to the buffer 1 h before the RNA extraction and 2% β-mercaptoethanol was added to the buffer just before use. The homogenate was incubated at 65°C for 5 min and extracted twice with chloroform: isoamylalcohol (24:1 v/v), each extraction followed by 10 min centrifugation at 5000 rpm and room temperature. RNA was precipitated overnight in 10 M LiCl at 4°C. After centrifugation at 10.000 rpm at 4°C for 20 min, the pellet was dissolved in SSTE buffer (1 M NaCl, 0.5% SDS, 10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8) and extracted with an equal volume of phenol (pH 4.5-5; Roti-Phenol, Roth A980): chloroform: isoamyl alcohol (25:24:1, v/v/v), followed by chloroform: isoamyl alcohol (24:1, v/v). Each extraction step was followed by 10 min centrifugation at 10000 rpm and 4°C. Two volumes of 100% ethanol were then added and RNA was precipitated for 2 hours at -20°C. After centrifugation for 20 min at 10000 rpm and 4°C, the pellet was washed with 80% ethanol, centrifuged 10 min at 10000 rpm and 4°C, air dried on ice, re-suspended in DEPC-treated water and quantified using Nanodrop 1000 (Thermo Scientifics, USA) and Qubit 2.0 (Life Technologies, Italy). RNA integrity was checked using a Bioanalyzer 2100 (Agilent Technologies, Italy).
cDNA libraries were prepared for sequencing using standard Illumina protocols by IGA Technology Services (Udine , Italy)
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
This sample is from mycorrhizal protocorm. It is the third of three biological replicates used in this experiment.
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| Data processing |
Illumina sofware was used by IGA to generate fastq raw data files
Reads were aligned to the Tulasnella calospora ( http://genome.jgi-psf.org/Tulca1 ) reference transcripts using CLC Genomics Workbench 6 and Reads Per Kilobase of exon per Megabase of library size (RPKM) were calculated.
Genome_build: Tulasnella calospora ( http://genome.jgi-psf.org/Tulca1 )
Supplementary_files_format_and_content: tab-delimited text files include transcript length,unique aligned reads, total aligned reads and RPKM values for each sample
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| Submission date |
Dec 04, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Annegret Kohler |
| E-mail(s) |
annegret.kohler@inrae.fr
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| Phone |
+33 (0)383 394072
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| Organization name |
INRAE
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| Department |
UMR 1136
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| Lab |
Interactions Arbres/Micro-organismes
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| Street address |
Centre INRAE Grand Est Nancy
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| City |
Champenoux |
| ZIP/Postal code |
54280 |
| Country |
France |
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| Platform ID |
GPL19502 |
| Series (2) |
| GSE63869 |
Gene expression changes in Tulasnella calospora- Serapias vomeracea mycorrhizal protocorms compared to Tulasnella calospora free-living mycelium |
| GSE63947 |
Transcriptome comparison of ectomycorrhizal, ericoid and orchid mycorrhizal fungi |
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| Relations |
| BioSample |
SAMN03252957 |
| SRA |
SRX796407 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1558695_p1.txt.gz |
214.2 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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