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| Status |
Public on Jun 20, 2014 |
| Title |
EXPRSS: an Illumina based high-throughput expression-profiling method to reveal transcriptional dynamics - III |
| Organism |
Arabidopsis thaliana |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
Background
Next Generation Sequencing technologies have facilitated differential gene expression analysis through RNA-seq and Tag-seq methods. RNA-seq has biases associated with transcript lengths, lacks uniform coverage of regions in mRNA and requires 10–20 times more reads than a typical Tag-seq. Most existing Tag-seq methods either have biases or not high throughput due to use of restriction enzymes or enzymatic manipulation of 5’ ends of mRNA or use of RNA ligations.
Results
We have developed EXpression Profiling through Randomly Sheared cDNA tag Sequencing (EXPRSS) that employs acoustic waves to randomly shear cDNA and generate sequence tags at a relatively defined position (~150-200 bp) from the 3′ end of each mRNA. Implementation of the method was verified through comparative analysis of expression data generated from EXPRSS, NlaIII-DGE and Affymetrix microarray and through qPCR quantification of selected genes. EXPRSS is a strand specific and restriction enzyme independent tag sequencing method that does not require cDNA length-based data transformations. EXPRSS is highly reproducible, is high-throughput and it also reveals alternative polyadenylation and polyadenylated antisense transcripts. It is cost-effective using barcoded multiplexing, avoids the biases of existing SAGE and derivative methods and can reveal polyadenylation position from paired-end sequencing.
Conclusions
EXPRSS Tag-seq provides sensitive and reliable gene expression data and enables high-throughput expression profiling with relatively simple downstream analysis.
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| Overall design |
Four-week-old Arabidopsis (No-0 and slh1) plants were grown at 28°C and transferred to a 21°C growth chamber. Time course samples were collected after the temperature shift; mRNA profiles were generated by deep sequencing on Illumina MiSeq using EXPRSS protocol.
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| Contributor(s) |
Rallapalli G, Kemen EM, MacLean D, Robert-Seilaniantz A, Segonzac C, Etherington G, Sohn KH, Jones JD |
| Citation(s) |
24884414 |
| |
| Submission date |
Nov 20, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Ghanasyam Rallapalli |
| E-mail(s) |
g.rallapalli@uea.ac.uk
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| Organization name |
The Sainsbury Laboratory
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| Street address |
Norwich Research Park
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| City |
Norwich |
| ZIP/Postal code |
NR4 7UH |
| Country |
United Kingdom |
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| Platforms (1) |
| GPL17970 |
Illumina MiSeq (Arabidopsis thaliana) |
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| Samples (9)
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| This SubSeries is part of SuperSeries: |
| GSE51721 |
EXPRSS: an Illumina based high-throughput expression-profiling method to reveal transcriptional dynamics |
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| Relations |
| BioProject |
PRJNA229430 |
| SRA |
SRP033221 |
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