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Sample GSM2322628 Query DataSets for GSM2322628
Status Public on Dec 01, 2016
Title Asymbiotic protocorm P2-3
Sample type SRA
 
Source name Asymbiotic protocorm
Organism Serapias vomeracea
Characteristics tissue: Asymbiotic, achlorophyllous protocorm stage P2
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.
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)
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2500
 
Description This sample is from asymbiotic, achlorophyllous protocorm. It is thethird of three biological replicates used in this experiment.
P2A-3
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
 
Submission date Sep 20, 2016
Last update date May 15, 2019
Contact name Annegret Kohler
E-mail(s) [email protected]
Phone +33 (0)383 394072
Organization name INRAE
Department UMR 1136
Lab Interactions Arbres/Micro-organismes
Street address Centre INRAE Grand Est Nancy
City Champenoux
ZIP/Postal code 54280
Country France
 
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
Relations
BioSample SAMN05791236
SRA SRX2178943

Supplementary file Size Download File type/resource
GSM2322628_P2A-3.txt.gz 1.7 Mb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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