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| Status |
Public on Jul 17, 2017 |
| Title |
At_pSUC2_TMV2 |
| Sample type |
SRA |
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| Source name |
At_pSUC2_TMV
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| Organism |
Arabidopsis thaliana |
| Characteristics |
ecotype: Shahdara
developmental stage: 5-week-old plants
genotype/variation: pSUC2:His6FLAG-RPL18
infected with: Tobacco mosaic virus
tissue: leaf phloem
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| Treatment protocol |
Plant leaves were inoculated with tobacco mosaic virus (1 mg/ml) or mock inoculated with sterile water. Inoculated leaves were harvested 6 days post infection and immediately frozen in liquid nitrogen.
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| Growth protocol |
Transgenic plants expressing promoter:His6FLAG-RPL18 (ecotype Shahdara) were maintained in growth chambers for a 12-h photoperiod, light 100 µmol s-1 m-2, at 24°C. Biological replicates were grown in different months under the same growth chamber conditions.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Frozen tissue was homogenized in Polysome Extraction Buffer (PEB; 200 mM Tris-HCl, pH 9.0, 200 mM KCl, 25 mM ethylene glycol tetraacetic acid (EGTA) pH 8.0, 35 mM MgCl2, 1% (v/v) octylphenyl-polyethylene glycol (Igepal CA-630), 1% (v/v) polyoxyethylene 10 tridecyl ether, 1% (v/v) sodium deoxycholate, 5 mM dithiothereitol (DTT), 1 mM PMSF, 50 ug/mL cycloheximide, 50 ug/mL chloramphenicol, 0.5 mg/mL heparin) using 10 mL PEB per 5 g of tissue. Homogenates were clarified by centrifugation at 16,000 g for 15 min and filtered with cheesecloth. Anti-FLAG magnetic beads, 50 μL, (Sigma Chemical Company, St. Louis, MO USA) were added to the clarified supernatant and incubated at 4oC overnight with gentle rocking. The beads were recovered using a magnet and washed four times for 5 min with 5 mL of wash buffer (200 mM Tris-HCl, pH 9.0, 200 mM KCl, 25 mM EGTA, 35 mM MgCl2, 5 mM DTT, 50 μg/mL cycloheximide, 50 μg/mL chloramphenicol). The complexes were eluted by treatment of the magnetic beads with 100 uL of elution buffer (100 uL wash buffer, 10 uL of 5mg/mL FLAG3 peptide - Sigma Chemical Company, St. Louis, MO USA, 0.5 uL of 2 U/mL RNAse OUT - Thermo Fisher Scientific Cleveland, Ohio, USA). RLT buffer plus 2-Mercaptoethanol from the Qiagen RNeasy kit (Qiagen, Valencia, CA USA) was added to the eluted complexes. A 0.5 X volume of 100% ethanol was then added and the sample transferred to Qiagen RNeasy columns. Washes and RNA elutions were performed according to the manufacturer’s instructions. Isolated RNA was measured on a NanoDrop 1000 and the quality of the RNA confirmed using a 2100 Bioanalyzer Eukaryotic Total RNA Nanochip (Agilent Technologies Palo Alto, CA USA).
cDNA libraries were prepared by the by the UM-IBBR Sequencing Core at University of Maryland, College Park (http://ibbr.umd.edu/facilities/sequencing) using the Illuminia TrueSeq RNA library kit according to the manufacturer's instructions.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 1500 |
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| Description |
polysomal RNA from leaf phloem tissue inoculated with tobacco mosaic virus
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| Data processing |
CASAVA 1.8.2 was used for bcl conversion and demultiplexing
Reads were mapped to the Arabidopsis thaliana Col-0 genome reference TAIR10 using the CLC Genomics Workbench v 7.5.1 RNA-seq analysis tool and default parameters. (Mismatch cost 2, Insertion cost 3, Deletion Cost 3, Length fraction 0.8, Similarity fraction 0.8, Max hits for a read 10)
Genome_build: Col-0 TAIR10
Supplementary_files_format_and_content: tab-delimited text files include Gene ID, Chromosome, Region, RPKM value, Total gene reads, and Total exon reads.
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| Submission date |
Apr 11, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
James N Culver |
| E-mail(s) |
[email protected]
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| Organization name |
University of Maryland, College Park
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| Department |
Plant Science and Landscape Architecture
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| Street address |
4291 Fieldhouse Dr.
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| City |
College Park |
| State/province |
MD |
| ZIP/Postal code |
20742 |
| Country |
USA |
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| Platform ID |
GPL19080 |
| Series (1) |
| GSE97662 |
Tobacco mosaic virus infection alters phloem associated translatomes in Arabidopsis thaliana |
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| Relations |
| BioSample |
SAMN06710692 |
| SRA |
SRX2733555 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2574842_At_pSUC2_TMV2.txt.gz |
519.2 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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