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Sample GSM1195627

Query DataSets for GSM1195627

Status

Public on Aug 15, 2014

Title

sRMyc1

Sample type

SRA

Source name

Root, treated with Myc-LCO factor

Organism

Medicago truncatula

Characteristics

tissue: Root

Treatment protocol

For the induction by both Myc-LCO and Nod factors, the A17 plants were obtained in the same way but with Fahraeus medium adjusted at 7.5 µM of phosphate. After five days, the plants were transferred in a 10-8 M Myc-LCO or Nod factors solution during 4 h and 24 h before being pooled dependently to the induction. For comparison between roots, root tips (1 cm long), plants of the Jemalong A17 genotype were cultivated in a hydroponic culture system on N-containing Fahräeus medium during 10 days at 23°C and 75% humidity (16-hour light/8-hour dark cycle). For the interaction with root pathogens, seedlings from the Jemalong A17 genotype, known to be susceptible to Ralstonia solanacearum GMI1000 strain and resistant to Verticillium albo-atrum V31.2 strain , were grown in an hydroponic culture system on N-containing Farhäeus medium in a growth chamber with 16 h light (170 µmol m–2 s–1) at 25 °C and 8 h dark at 23 °C. Inoculation with V. albo-atrum was performed as described in Ben C. Journal of experimental botany 2013. Inoculated plants were then transferred back to the nutritive solution and incubated in a growth chamber at 20 °C with 16 h photoperiod. For inoculation with R. solanacearum, cut roots were immersed for 30 min in a bacterial solution of the GMI1000 strain at a concentration of 108CFU/ml. Inoculated plants and controls treated with water were then placed in a phytotron with 12 h light (170 µmol m–2 s–1) at 28 °C and 12 h dark at 26 °C. For each condition of inoculation, samples were obtained as pools of roots of five to fifty plants harvested every day from one day to one week after inoculation.For the interaction with Rhizophagus irregularis (DAOM197198), plants from the A17 line were grown in a greenhouse (16-hour light/8-hour dark cycle) for 7 weeks in a soil substrate inoculated with 1000 spores/liters. The substrate was hydrated with a Long-Ashton solution with a phosphate concentration of 7.5 µM. The well-established mycorrhization was controlled by the grid intersection method. For the interaction with Sinorhizobium meliloti plants from the sunn-sickle hypernodulating double mutant were grown in an aeroponic chamber at 23 °C and 75 % of humidity during five days (16-hour light/8-hour dark cycle) in a nitrogen-less liquid Fahraeus medium nebulization inoculated with bacteria .

Growth protocol

Plants from the reference sequenced line Jemalong A17 or from the sunn-sickle hypernodulating double mutant (Jemalong A17 genetic background, of the barrel medic (Medicago truncatula L.)) were grown in different way depending on the interaction of interest. For the interaction with Rhizophagus irregularis (DAOM197198), plants from the A17 line were grown in a greenhouse (16-hour light/8-hour dark cycle) for 7 weeks in a soil substrate inoculated with 1000 spores/liters. The substrate was hydrated with a Long-Ashton solution with a phosphate concentration of 7.5 µM. The well-established mycorrhization was controlled by the grid intersection method. For the interaction with Sinorhizobium meliloti plants from the sunn-sickle hypernodulating double mutant were grown in an aeroponic chamber at 23 °C and 75 % of humidity during five days (16-hour light/8-hour dark cycle) in a nitrogen-less liquid Fahraeus medium nebulization inoculated with bacteria . For the induction by both Myc-LCO and Nod factors, the A17 plants were obtained in the same way but with Fahraeus medium adjusted at 7.5 µM of phosphate. After five days, the plants were transferred in a 10-8 M Myc-LCO or Nod factors solution during 4 h and 24 h before being pooled dependently to the induction. For comparison between roots, root tips (1 cm long), plants of the Jemalong A17 genotype were cultivated in a hydroponic culture system on N-containing Fahräeus medium during 10 days at 23°C and 75% humidity (16-hour light/8-hour dark cycle). For the interaction with root pathogens, seedlings from the Jemalong A17 genotype, known to be susceptible to Ralstonia solanacearum GMI1000 strain and resistant to Verticillium albo-atrum V31.2 strain, were grown in an hydroponic culture system on N-containing Farhäeus medium in a growth chamber with 16 h light (170 µmol m–2 s–1) at 25 °C and 8 h dark at 23 °C. Inoculation with V. albo-atrum was performed as described in paper Ben C. Journal of experimental botany 2013. Inoculated plants were then transferred back to the nutritive solution and incubated in a growth chamber at 20 °C with 16 h photoperiod. For inoculation with R. solanacearum, cut roots were immersed for 30 min in a bacterial solution of the GMI1000 strain at a concentration of 108CFU/ml. Inoculated plants and controls treated with water were then placed in a phytotron with 12 h light (170 µmol m–2 s–1) at 28 °C and 12 h dark at 26 °C. For each condition of inoculation, samples were obtained as pools of roots of five to fifty plants harvested every day from one day to one week after inoculation.

Extracted molecule

total RNA

Extraction protocol

Plant roots were harvested and crushed with liquid nitrogen. For the Myc-LCO and Nod factor libraries and their corresponding controls, total RNA were isolated from 100 mg of powder with the mirVana miRNA isolation kit (Ambion) according to the manufacturer’s instructions. For the other libraries, total RNAs were extracted using 1 mL of Trizol per 50-100 mg of powder. Phenol-Chloroform separation, isopropanol precipitation, ethanol wash and RNA solubilization were performed according to the TRI REAGENT manufacturer’s instructions. RNA integrity and quality have been checked thanks to the Agilent Bioanalyzer 2100.
The smRNA libraries were obtained thanks to the Small RNA Sample Prep kit from Illumina according to the “Preparing Samples for Small RNA sequencing using the Alternative v1.5” protocol. libraries were sequenced using HiSeq Solexa sequencer from Illumina.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

size fractionation

Instrument model

Illumina HiSeq 2000

Description

small RNA fraction

Data processing

Raw reads of smRNA sequencing were filtered by the LeARN Pipeline adapted for plant smRNAs. In brief, reads were cleaned (adaptors suppression, 18-25nt size filtering, reads containing ‘N’ suppression and suppression of tRNA and rRNA), the redundancy was counted and removed.
Supplementary_files_format_and_content: Multifasta file of cleaned smRNA. The fasta headers give the number of times each sequence is present in each library

Submission date

Jul 25, 2013

Last update date

May 15, 2019

Contact name

Erika Sallet

E-mail(s)

[email protected]

Organization name

INRA/CNRS

Department

Plant Health and the Environment