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| Status |
Public on Sep 02, 2017 |
| Title |
35S:RPS4, T0, rep2 |
| Sample type |
SRA |
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| Source name |
35S:RPS4, T0
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| Organism |
Arabidopsis thaliana |
| Characteristics |
tissue: leaves
ecotype/cultivar: Col-0
genotype: 35S:RPS4-HS
age: 3,5 week-old
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| Treatment protocol |
Plants were shifted from 28°C to 19°C for 4 and 8h before collecting and freezing the samples. T0 samples were not shifted.
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| Growth protocol |
Plants were grown for 3,5 weeks at 28°C
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA were extracted using the Qiagen RNeasy kit. RNA were DNaseI-treated and a purification was done using the Qiagen RNeasy MinElute kit. Ribosomal RNAs were depleted from 2.5 µg RNA using the RiboMinus Plant Kit for RNA-seq (Life Technologies). For RNA-seq library preparation, we followed the Illumina "TruSeq RNA Sample Preparation v2 Guide" starting with poly-A enrichment followed by standard cDNA synthesis, end repair, A-tailing, adapter ligation and PCR enrichment (15 cycles). Libraries were quantified by fluorometry (Picogreen, Invitrogen) and equimolar mixtures of libraries were loaded onto the cBot station (Illumina) for cluster generation prior to sequencing on a HiSeq2000.
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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 |
Illumina CASAVA1.8 software was used for base calling.
Sequenced paired-end reads were separately clipped for adapter sequence and trimmed for low quality retaining reads with average Phred+33 quality/read >= 30 and length >= 50 bases. Reads that did not match these criteria were discarded.
The adapter clipped quality trimmed reads were then balanced for those cases where both the pairs passed filtering. In those cases where only one pair passed filtering, they were written on to a separate single-end file with the idea of mapping them separately in order to consider those reads as they passed filtering.
Reads were mapped on to Arabidopsis thaliana TAIR10 genome using tophat version 2.0.0 with parameters -m 0 -g 1 --genome-read-mismatches 2 --read-mismatches 2 --library-type fr-unstranded.
mean inner distance and standard deviation for paired end reads were estimated separately for each sample using a custom made perl script by first mapping the reads preliminarily to Arabidopsis thaliana genome using BWA version 0.5.9 and then calculating from the bam file mean and standard deviation of inner distance between pairs that fall in the range [Q1 - 2*(Q3-Q1), Q3 + 2*(Q3-Q1)], where Q1 and Q3 are the first and the third quartiles. The junctions.bed file from the paired-end run was converted to a junctions file using bed_to_juncs script that comes with tophat and fed to the SE reads that were generated with the -j parameter for the second run.
Genome_build: TAIR 10
Supplementary_files_format_and_content: Tab delimited text files of read counts across junctions (introns) with the type of splicing event they fall to - 1) NJ (Normal junction - intron as found in the first transcript), 2) ES (Exon skipping), 3) A3P(Alternative 3prime), 4) A5P(Alternative 5prime), 5) IR(Intron retention), 6) CI(Cryptic intron).
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| Submission date |
Aug 20, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Servane Baufume |
| Organization name |
Max Planck Institute for Plant Breeding Research
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| Department |
Plant Microbe Intreractions
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| Lab |
Parker
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| Street address |
Carl-von-Linné-Weg 10
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| City |
Koeln |
| State/province |
Germany (DEU) |
| ZIP/Postal code |
50829 |
| Country |
Germany |
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| Platform ID |
GPL13222 |
| Series (1) |
| GSE40216 |
Study of expression changes and alternative splicing events during RPS4-mediated resistance in Arabidopsis using a temperature-inducible system which mirrors pathogen-infection |
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| Relations |
| SRA |
SRX179392 |
| BioSample |
SAMN01120981 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM988488_RPS4_T0_REP2.txt.gz |
1.3 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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