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Status |
Public on Jan 01, 2018 |
Title |
Free-living mycelium-3 |
Sample type |
SRA |
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Source name |
Free-living mycelium
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Organism |
Hyaloscypha bicolor |
Characteristics |
tissue: free-living mycelium
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Growth protocol |
Meliniomyces bicolor was grown on a modified MMN medium containing KH2PO4 0.5 gL-1, BSA (bovine serum albumin, SIGMA) 0.1 gL-1, CaCl2*2H2O 0.066 gL-1, NaCl 0.025 gL-1, MgSO4*7H2O 0.15 gL-1, thiamine-HCl 0.1 gL-1, FeCl3*6H2O 0.001 gL- 1, agar 10 gL-1. BSA and thiamine-HCl were added to the medium as filter sterilized solutions just before plating. The final pH was 4.7. Prior to fungal inoculation, sterile cellophane membranes were placed aseptically on the agar surface to provide a convenient means of removing the mycelium from the plate. Plates (15 cm in diameter, 2.5 cm in height) were inoculated with 5 mm fungal plugs. Fungal colonies were removed after 113 days by peeling the biomass from the cellophane membrane. Several plates were prepared in order to get enough fungal material to perform three separate RNA extractions for the RNASeq experiment. Endomycorrhizae were synthesized aseptically in plastic petri plates (15 cm in diameter, 2.5 cm in height) containing the same medium reported above for fungal cultures. Control, non-inoculated Vaccinium myrtillus plants were grown on the same medium but (NH4)2HPO4 0,075 gL-1 was added instead of the BSA. Axenic V. myrtillus seedlings were obtained from surfacesterilized (70% ethanol, vol/vol, plus 0.2% Tween 20 for 3 min; rinsed twice with sterile water and 0.25% sodium hypochlorite for 15 min, with three additional sterile water rinses) bilberry seeds (obtained by Les Semences du Puy, Le Puy-En-Velay, France) germinated on water and 1% agar petri plates in the dark for 2 weeks before transfer to the growth chamber for 1 month. A conidia suspension in sterile deionised water was prepared from 1 month-old fungal cultures grown on Czapek-glucose solid medium (NaNO3 3 g L-1, K2HPO4*3H2O 1.31 g L-1, MgSO4*7H2O 0.5 g L-1, FeSO4*7H2O 0.01 g L-1, KCl 0.5 g L-1, glucose 20 gL-1, agar 10 gL-1). All reagents were purchased from Sigma. The medium was adjusted to pH 6 with the addition of 1M HCl. The conidia suspension was distributed in the bottom half of the MMN petri plates. Ten germinated V. myrtillus seedlings were then transferred aseptically in the MMN plates at the upper limit of the surface covered by the conidia suspension. Finally the plates were sealed and placed in a growth chamber (16-h photoperiod, light at 170 μmol m–2 s–1, temperatures at 23°C day and 21°C night). Several plates were prepared in order to get enough root material to perform three separate RNA extractions for the RNASeq experiment. The root systems were observed after 113 days incubation period and the percentage of mycorrhization was evaluated. Prior the observation, each Vaccinium hair-root system from the synthesis plates was stained in 0.1 % (wt/vol) cotton blue (methyl blue) overnight and distained with 80% lactic acid. Whole roots were then mounted in the distaining solution, observed using a Nikon Eclipse E400 optical microscope, and photographed.
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Extracted molecule |
total RNA |
Extraction protocol |
RNA was extracted from M. bicolor mycelia and from M. bicolor-inoculated V. myrtillus roots 113 days after cultures start. 100 mg aliquots of mycelium were collected, frozen in liquid nitrogen, mechanically ground 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) extraction step and a chloroform extraction step followed. Each extraction step was followed by 5 min centrifugation at 14000 rpm and 4°C. A isopropyl alcohol precipitation step at -80°C for 30 min was than performed followed by 30 min centrifugation at 14000 rpm and 4°C. The pellet was then re-suspended in a 1:1 DEPC treated water:6M LiCl solution and precipitated over-night 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 the nanodrop and the bioanalyzer. Total RNA was extracted from V. myrtillus mycorrhizal roots using the CTAB method. 100 mg aliquots of V. myrtillus mycorrhizal roots were collected, frozen in liquid nitrogen, mechanically ground and RNA was extracted in warm (65°C) CTAB buffer (2% CTAB, 2% PVP, 100 mM Tris-HCl pH 8.0; 25 mM EDTA pH 8; 2 M NaCl). 2% PVPP was added to the buffer 1 h before performing the extraction letting it to get hydrated and 2% β-mercaptoethanol was added to the buffer just before use. The extraction suspension was incubated at 65°C for 5 min. Two chloroform/isoamylalcohol (24:1) extraction steps followed. Each extraction step was followed by 10 min centrifugation at 5000 rpm and room temperature. An overnight precipitation step in 10 M LiCl at 4°C followed. After a centrifugation at 10.000 rpm 4°C for 20 min, pellet was dissolved in SSTE buffer (1 M NaCl, 0.5% SDS, 10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8). Extraction steps with an equal volume of 1:1 phenol (pH 4.5-5; Roti-Phenol, Roth A980):chloroform/isoamyl alcohol (24:1) and of chloroform/isoamyl alcohol (24:1) followed. Each extraction step was followed by 10 min centrifugation at 10000 rpm and 4°C. Two volumes of ethanol 100% were then added and a precipitation step of 2 hours at -20°C followed. After a centrifugation of 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 the nanodrop and the bioanalyzer. cDNA libraries were prepared for sequencing using standard Illumina protocols by the Joint Genome Institue (JGI)
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 2500 |
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Description |
This sample is from free-living mycelium. It is the third of three biological replicates used in this experiment.
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Data processing |
Illumina sofware was used by the JGI to generate fastq raw data files Reads were aligned to Meliniomyces bicolor (https://genome.jgi.doe.gov/Melbi2/Melbi2.home.html) reference transcripts using CLC Genomics Workbench 8 and Reads Per Kilobase of exon per Megabase of library size (RPKM) were calculated. Genome_build: Meliniomyces bicolor (https://genome.jgi.doe.gov/Melbi2/Melbi2.home.html) Supplementary_files_format_and_content: tab-delimited text files include unique aligned reads, total aligned reads and RPKM values for each sample
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Submission date |
Dec 08, 2017 |
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 |
GPL24356 |
Series (1) |
GSE107845 |
Gene expression changes in Meliniomyces bicolor- Vaccinium myrtillus mycorrhizal roots compared to Meliniomyces bicolor free-living mycelium |
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Relations |
BioSample |
SAMN08150310 |
SRA |
SRX3459066 |
Supplementary file |
Size |
Download |
File type/resource |
GSM2881534_MbFLM-3.txt.gz |
223.6 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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