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| Status |
Public on Jul 06, 2016 |
| Title |
G2_drought stress_Desiree |
| Sample type |
SRA |
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| Source name |
terminal leaflet of the first mature source leaf
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| Organism |
Solanum tuberosum |
| Characteristics |
tissue: leaf
experiment: G2
treatment: drought stress
cultivar: Desiree
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| Treatment protocol |
Control plants were irrigated optimally during the whole experiment, whereas drought stress was induced by withholding water. Regarding greenhouse (G) experiments, the drought stress experiment started twelve days after transfer to the final pot size by stopping the irrigation of the plants in the drought stress blocks. Plants of the control block were watered by drip-irrigation twice to thrice a week with the amount of water lost by evapotranspiration since the last irrigation event determined by weighing pots. From day 20 onwards, plants in the drought stress blocks received 30% of the amount of water given to the control plants. From day 60 onwards, drought stressed plants received 45% of the amount given to the control plants (G1+2) or furthermore 30% (G3 ). In F1 and F3 plants were drip-irrigated from the top of the ridges with 10mm water during the night when plants in control plots showed signs of decreased turgor at noon. Drought stressed plants were irrigated when they showed signs of water stress 2 h after sunrise. F4 was carried out in a rain-out-shelter where drought stress was applied by stopping watering at the beginning of emergence.
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| Growth protocol |
Two greenhouse (G) experiments were conducted in a climate-controlled greenhouse cabin at the MPI-MP, Potsdam, Germany (G1, G2) while experiment G3 was conducted in a greenhouse at the JKI Groß Lüsewitz, Germany. Pot experiments were started from axenically grown plants that had been propagated at 22°C and 16 h light at the MPI-MP and at 20°C (light)/17°C (dark) at the JKI. For G1 and G2, plantlets were transferred from in vitro cultivation to pots with pricking substrate and acclimated for a week in a climate chamber at 22°C/70% RH during the 16 h day with a light intensity of 150 µmol m-2s-1 and 18°C/70% RH during the night. Subsequently, plants were transferred to a climate-controlled greenhouse chamber with 20°C/60% RH during the 16 h day and 18°C/50% RH during the night. After another week, plants were transferred to the final 18 cm diameter pots. In G3, in vitro plants were transferred to pots in a greenhouse and irrigated as required. After 14 days, plants were transferred to pots with a volume of 5 l and pots were moved to the rain-out-shelter. Two field (F) experiments were conducted at the MPI-MP, Potsdam, Germany (F1, F3) while experiment F4 was conducted at the JKI Groß Lüsewitz, Germany. For F1 and F3, tubers were pre-sprouted at 8-10°C and planted into ridges at a depth of approximately 15-20 cm, with a spacing of 30 cm within a row and 75 cm between rows. Plants were sprayed twice with magnesium sulphate solution (25 kg/ha) to compensate for low Mg availability in the soil. Potatoes were sprayed with an insecticide against Colorado beetle larvae when the peak of young larvae was observed. F4 was carried out under a rain-out-shelter and tubers were planted in a split-plot design with two watering regimes. The control plots were cultivated next to the shelter under natural rainfall conditions plus additional irrigation.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Homogenized leaf material of two to four replicates per cultivar and treatment was pooled to approximate 100 mg. Total RNA was isolated using a TRIzol protocol based on the “single step” method (Chomczynski and Sacchi 1987). For extraction 1 mL TRIzol reagent (Invitrogen) and 200 µL chloroform were used. The supernatant was precipitated by adding 500 µL isopropanol and 250 µL 2 M NaCl. After washing the pellet twice with 70% ethanol, it was re-suspended in 50 µL RNase-free water.
Samples were transferred to the BGI in China for cDNA library preparation and sequencing. The total RNA samples are first treated with DNase I. Then the mRNA is enriched by using the oligo(dT) magnetic beads. Mixed with the fragmentation buffer, the mRNA is fragmented into short fragments. Then the first strand of cDNA is synthesized by using random hexamer-primer. Buffer, dNTPs, RNase H and DNA polymerase I are added to synthesize the second strand. The double strand cDNA is purified with magnetic beads. End reparation and 3'-end single nucleotide A (adenine) addition is then performed. Finally, sequencing adaptors are ligated to the fragments. The fragments are enriched by PCR amplification. During the QC step, Agilent 2100 Bioanaylzer and ABI StepOnePlus Real-Time PCR System are used to qualify and quantify of the sample library. The library products are ready for sequencing via Illumina HiSeqTM 2000.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Quality of pre-filtered read sequences was tested by FastQC version 0.11.2 (Babraham Institute, http://www.bioinformatics.babraham.ac.uk/projects/fastq). As no overrepresented sequences were detected, an additional 3’-trimming step was skipped.
Mapping was performed by RSEM v1.2.3 using default parameters and its recommended mapper bowtie v.0.12.9. EnsemblPlants release 17 of Genomic FASTA and cDNA GTF file of S. tuberosum were used to obtain a cDNA fasta file with a 125bp long poly-A tail per transcript generated by RSEM module rsem-prepare-reference.
The RSEM module rsem-calculate-expression outputs transcript abundance measurements as expected counts, Transcripts Per Million reads (TPM) and Fragments Per Kilobase of Exon Per Million reads (FPKM).
Genome_build: EnsemblPlants version 17 of Solanum tuberosum
Supplementary_files_format_and_content: Default RSEM output files (ASCII format). Columns: Gene id, Transcript id, length, effective_length, expected_count, TPM, FPKM
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| Submission date |
Feb 11, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Ellen Zuther |
| E-mail(s) |
[email protected]
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| Phone |
+49 331 567 8253
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| Organization name |
MPI of Molecular Plant Physiology
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| Department |
Transcript Profiling
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| Street address |
Am Muehlenberg 1
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| City |
Potsdam |
| ZIP/Postal code |
D-14476 |
| Country |
Germany |
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| Platform ID |
GPL16436 |
| Series (1) |
| GSE77826 |
Metabolic and transcriptomic responses of leaves from European potato reference cultivars with differential tolerance to long-term drought |
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| Relations |
| SRA |
SRX1571291 |
| BioSample |
SAMN04501484 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2060138_sample30.genes.results.txt.gz |
696.5 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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