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| Status |
Public on Aug 03, 2015 |
| Title |
WT Col control rep 3 |
| Sample type |
SRA |
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| Source name |
pool of tissues
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| Organism |
Arabidopsis thaliana |
| Characteristics |
ecotype: Col
genotype: Wild type
tissue: whole plant
age (days): 9
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| Treatment protocol |
Whole plants were harvested after 9 d.
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| Growth protocol |
WT (Col) and gemin2 mutants seeds were grown on Murashige and Skoog medium containing 0.8% agarose, stratified for 4 d in the dark at 4 °C, and then grown for nine days under continuous white light at 22ºC or exposed for 1 or 24 h to 10ºC on the 9th day, before harvesting.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted with RNeasy Plant Mini Kit (QIAGEN) following the manufacturer’s protocols. To estimate the concentration and quality of samples, NanoDrop 2000c (Thermo Scientific) and gel electrophoresis were used, respectively.
Libraries were prepared following the TruSeq RNA Sample Preparation Guide (Illumina). Briefly, 3 μg of total RNA was polyA-purified and fragmented, and first-strand cDNA synthesized by reverse transcriptase (SuperScript II; Invitrogen) and random hexamers. This was followed by RNA degradation and second-strand cDNA synthesis. End repair process and addition of a single A nucleotide to the 3′ ends allowed ligation of multiple indexing adapters. Then, an enrichment step of 12 cycles of PCR was performed. Library validation included size and purity assessment with the Agilent 2100 Bioanalyzer and the Agilent DNA1000 kit (Agilent Technologies)
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 1500 |
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| Data processing |
Basecalling done with Bustard Illumina Pipelline
Sequence reads were mapped to Arabidopsis thaliana TAIR10 genome using TopHat v2.0.9 with default parameters, except of maximum intron length set in 5000
Count tables for different feature levels were obtained from bam files using custom R v3.0.2 scripts.
Differential gene expression was estimated using edgeR package version 3.4.2 and resulting p-values were adjusted using false discovery rate (fdr) criterion.
The transcriptome was partitioned into subgenic joint features called bins and labeled as exon-bin, intron-bin or alternative-splicing (AS) bins. In addition AS-bins were further classified as exon skipping (ES), 5’ or 3’ alternative (5´alt, 3´alt), intron retention (IR) or multiple. Bins from monoexonic genes and with mean count values lower than 5 reads per condition were discarded.
We used edgeR exactTest for the identification of differential usage of bins corresponding to AS events or introns, and fdr corrected p-values. We also computed read densities in order to have a relationship between the bin and its corresponding gene. A Splicing Index was calculated as bin read density/gene read density, and the Splicing Index Ratio was calculated as Splicing Index in mutants/Splicing Index in WT plants. Only genes with read densities greater than 0.1 in all genotypes, and Splicing Indexes greater than 0.05 in at least one genotype, were used for the analysis. AS events as well as all introns with an absolute Log2 FC (bin read density in the mutant/bin read density in WT) value greater than 0.58, with fdr values lower than 0.15, and an abosolute Log2 Splicing Index Ratio (Splicing Index in the mutant/Splicing Index in WT) greater than 0.58, were deemed as differentially spliced.
Genome_build: Arabidopsis thaliana TAIR10
Supplementary_files_format_and_content: Comprehensive spreadsheets with the results of the analysis of differential expression and differential splicing detailed above
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| Submission date |
Nov 18, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Marcelo Yanovsky |
| E-mail(s) |
myanovsky@leloir.org.ar
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| Organization name |
Fundación Instituto Leloir. IIBBA- CONICET
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| Lab |
Laboratorio de Genómica Comparativa del Desarrollo Vegetal
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| Street address |
Av. Patricias Argentinas 435
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| City |
Buenos Aires |
| State/province |
Ciudad Autonoma de Buenos Aires |
| ZIP/Postal code |
C1405BWE |
| Country |
Argentina |
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| Platform ID |
GPL19080 |
| Series (2) |
| GSE63406 |
Genome-wide analysis of wild type and gemin2 mutant plants [cold exposure] |
| GSE63407 |
Genome-wide analysis of wild type and gemin2 mutant plants exposed to cold |
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| Relations |
| BioSample |
SAMN03199302 |
| SRA |
SRX761597 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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