Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

High-throughput pyrosequencing of endogenous small RNAs from Solanum lycopersicum leaf and fruit

ABSTRACT: Comparison of the endogenous small RNA content of tomato leaves and fruits. Size fractionated small RNA from total RNA extracts was ligated to adapters, purified again and reverse transcribed. After PCR amplification the sample was subjected to 454 high throughput pyrosequencing. Please see www.454.com for details of the sequencing technology. Note: Raw data files were not available from 454 at the time this experiment was carried out.

ORGANISM(S): Solanum lycopersicum

SUBMITTER: Frank Schwach

PROVIDER: E-GEOD-12081 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Deep sequencing of tomato short RNAs identifies microRNAs targeting genes involved in fruit ripening.

Moxon Simon S Jing Runchun R Szittya Gyorgy G Schwach Frank F Rusholme Pilcher Rachel L RL Moulton Vincent V Dalmay Tamas T

Genome research 20080724 10

In plants there are several classes of 21-24-nt short RNAs that regulate gene expression. The most conserved class is the microRNAs (miRNAs), although some miRNAs are found only in specific species. We used high-throughput pyrosequencing to identify conserved and nonconserved miRNAs and other short RNAs in tomato fruit and leaf. Several conserved miRNAs showed tissue-specific expression, which, combined with target gene validation results, suggests that miRNAs may play a role in fleshy fruit dev ...[more]

PMID: 18653800

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Project description:We profiled vsiRNAs using two different high-throughput sequencing platforms and also developed a hybridisation-based array approach. The profiles obtained through the Solexa platform and by hybridisation were very similar to each other but different from the 454 profile. Both deep sequencing techniques revealed a strong bias in vsiRNAs for the positive strand of the virus and identified regions on the viral genome that produced vsiRNA in much higher abundance than other regions. The hybridisation approach also showed that the position of highly abundant vsiRNAs were the same in different plant species and in the absence of RDR6. We used the TerminatorTM 5'-Phosphate-Dependent Exonuclease to study the 5' end of vsiRNAs and showed that a perfect control duplex was not digested by the enzyme without denaturation and that the efficiency of the Terminator was strongly affected by the concentration of the substrate. We found that most vsiRNAs have 5' monophosphates, which was also confirmed by profiling short RNA libraries following either direct ligation of adapters to the 5' end of short RNAs or after replacing any potential 5' ends with monophosphates. The Terminator experiments also showed that vsiRNAs were not perfect duplexes. Using a sensor construct we also found that regions from the viral genome that were not complementary to highly abundant vsiRNAs were targeted in planta just as efficiently as regions recognised by abundant vsiRNAs. Different high-throughput sequencing techniques have different reproducible sequence bias and generate different profiles of short RNAs. The Terminator exonuclease does not process double-stranded RNA and because short RNAs can quickly re-anneal at high concentration, this assay can be misleading if the substrate is not denatured and not analysed in a dilution series. The sequence profiles and Terminator digests suggest that CymRSV siRNAs are produced from the structured positive strand rather than from perfect double-stranded RNA or by RNA-dependent RNA polymerase. Size-fractionated (19-24 nt) small RNA from early systemic leaves of CymRSV-infected Nicotiana benthamiana total RNA extracts was ligated to adapters, purified again and reverse transcribed. After PCR amplification, the sample was subjected to 454 high throughput pyrosequencing. Please see www.454.com for details of the sequencing technology.

Dataset Information

High-throughput pyrosequencing of endogenous small RNAs from Solanum lycopersicum leaf and fruit

Publications

Deep sequencing of tomato short RNAs identifies microRNAs targeting genes involved in fruit ripening.

Similar Datasets

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