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| Status |
Public on Dec 01, 2016 |
| Title |
Asymbiotic protocorm P2-2 |
| Sample type |
SRA |
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| Source name |
Asymbiotic protocorm
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| Organism |
Serapias vomeracea |
| Characteristics |
tissue: Asymbiotic, achlorophyllous protocorm stage P2
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| Growth protocol |
Symbiotic germination was obtained by co-inoculation of mycorrhizal fungi and orchid seeds in 9 cm Petri dishes. Seeds of S. vomeracea were surface sterilized in 1% sodium hypochlorite and 0.1% Tween-20 for 20 minutes on a vortex, followed by three 5-minutes rinses in sterile distilled water. Seeds were re-suspended in sterile water and dropped on strips of autoclaved filter paper (1.5 x 3 cm) positioned on solid oat medium (0.3% milled oats, 1% agar). Plates were inoculated with a plug of actively growing T. calospora mycelium and were incubated at 20°C in full darkness. Asymbiotic seed germination was obtained on modified BM1 culture media at 20°C in darkness. Symbiotic and asymbiotic achlorophyllous protocorms collected at stage P2 were frozen immediately in liquid nitrogen and stored at -80 °C for RNA extraction.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using the CTAB method. 100 mg aliquots 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 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 asymbiotic, achlorophyllous protocorm. It is the second of three biological replicates used in this experiment.
P2A-2
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| Data processing |
Illumina sofware was used by the JGI to generate fastq raw data files
Reads were aligned to Serapias vomeracea reference contigs using CLC Genomics Workbench 9 and Reads Per Kilobase of exon per Megabase of library size (RPKM) were calculated.
Genome_build: Serapias vomeracea reference (de novo transcript assembly)
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 |
Sep 20, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Annegret Kohler |
| E-mail(s) |
[email protected]
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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 |
GPL22451 |
| Series (1) |
| GSE87120 |
Gene expression changes in Tulasnella calospora - Serapias vomeracea mycorrhizal protocorms at stage P2 compared to asymbiotic, achlorophyllous Serapias vomeracea protocorms |
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| Relations |
| BioSample |
SAMN05791237 |
| SRA |
SRX2178942 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2322627_P2A-2.txt.gz |
1.4 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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