Project description:Vitis vinifera endogenous small RNAs Size fractionated small RNA from total RNA extracts of Vitis vinifera leaves, inflorescences, tendrils and small berries were ligated to adapters, purified again and reverse transcribed. After PCR amplification the sample was subjected to Solexa/Illumina high throughput pyrosequencing. Please see www.illumina.com for details of the sequencing technology.
Project description:Size fractionated small RNA from total RNA extracts of Vitis vinifera leaves were ligated to adapters, purified again and reverse transcribed. After PCR amplification the sample was subjected to Illumina high throughput pyrosequencing. The kit used is TrueSeq Small RNA kit Please see www.illumina.com for details of the sequencing technology.
Project description:Vitis vinifera RNA degradome Isolated polyadenylated RNA from total RNA extracts of Vitis vinifera leaves, were ligated to 5'-adapter that include san MmeI recognition site. The ligated products were purified again, reverse transcribed and cleaved with MmeI. The 5' fragments were purified from gel and to a 3'- dsDNA adapter and PCR amplified. After PCR amplification the sample was subjected to Solexa/Illumina high throughput pyrosequencing. Please see www.illumina.com for details of the sequencing technology.
Project description:Small RNAs (21-24 nt) are pivotal regulators of gene expression that guide both transcriptional and post-transcriptional silencing mechanisms in diverse eukaryotes, including most if not all plants. MicroRNAs (miRNAs) and short interfering RNAs (siRNAs) are the two major types, both of which have a demonstrated and important role in plant development, stress responses and pathogen resistance. In this work, we used a deep sequencing approach (Sequencing-By-Synthesis, or SBS) to develop sequence resources of small RNAs from Vitis vinifera tissues (including leaves, flowers and berries). The high depth of the resulting datasets enabled us to examine in detail critical small RNA features as size distribution, tissue-specific regulation and sequence conservation between different organs in this species. We also developed database resources and a dedicated website (http://smallrna.udel.edu/) with computational tools for allowing other users to identify new miRNAs or siRNAs involved in specific regulatory pathways, verify the degree of conservation of these sequences in other plant species and map small RNAs on genes or larger regions of the genome under study. Small RNA libraries were derived from leaves flowers and berries of Vitis vinifera. Total RNA was isolated using the Plant RNA Purification Reagent (Invitrogen), and submitted to Illumina (Hayward, CA, http://www.illumina.com) for small RNA library construction using approaches described in (Lu et al., 2007) with minor modifications. The small RNA libraries were sequenced with the Sequencing-By-Synthesis (SBS) technology by Illumina. PERL scripts were designed to remove the adapter sequences and determine the abundance of each distinct small RNA. We thank Gabriele Di Gaspero for providing the plant material as well as Kan Nobuta and Gayathri Mahalingam for assistance with the computational methods.
Project description:Small RNAs (21-24 nt) are pivotal regulators of gene expression that guide both transcriptional and post-transcriptional silencing mechanisms in diverse eukaryotes, including most if not all plants. MicroRNAs (miRNAs) and short interfering RNAs (siRNAs) are the two major types, both of which have a demonstrated and important role in plant development, stress responses and pathogen resistance. In this work, we used a deep sequencing approach (Sequencing-By-Synthesis, or SBS) to develop sequence resources of small RNAs from Vitis vinifera tissues (including leaves, flowers and berries). The high depth of the resulting datasets enabled us to examine in detail critical small RNA features as size distribution, tissue-specific regulation and sequence conservation between different organs in this species. We also developed database resources and a dedicated website (http://smallrna.udel.edu/) with computational tools for allowing other users to identify new miRNAs or siRNAs involved in specific regulatory pathways, verify the degree of conservation of these sequences in other plant species and map small RNAs on genes or larger regions of the genome under study.
Project description:In the present study we have generated a parallel analysis of the 5' RNA ends (PARE) referred to polyadenylated RNAs in somatic embryos from in vitro culture of immature anthers of Vitis vinifera. PARE analysis include also a shortRNA analysis of same material. We have submitted the sRNAseq to mirPROOF and miRCAT (srna_workbench software at http://srna-workbench.cmp.uea.ac.uk) in order to recognize known and putative novel miRNAs, respectively that could be present in somatic embryos. In addition, aRNAs and PARE libraries were integrated in order to identify those 5' RNA ends that could be explained by sRNAs (i.e. identify transcripts that could be cleaved by sRNAs). We have used publicly available annotations and we gained a deep insight into the gene families and the transcriptional regulation mediated by miRNAs in V. vinifera somatic embryos. Gene expression is finely regulated to specific paralogues in gene families such as the NADPH-dependent diflavin oxidoreductase, Phosphoserine aminotransferase, Ethylene-responsive transcription factors, glutathione S-transferase par C, just to name a few. We have indeed characterized at least 4,000 mRNA targets. Size fractionated small RNA from total RNA extracts of somatic Embryos cv. "Brachetto" were ligated to adapters, purified again and reverse transcribed. After PCR amplification the sample was subjected to Illumina high throughput pyrosequencing. The kit used is TrueSeq Small RNA kit, Illumina. Please see www.illumina.com for details of the sequencing technology. PARE libraries were generated starting from Poly(A) fraction. The protocol used have been previously described in German et al., 2009, Nature protocols. Please see www.illumina.com for details of the sequencing technology.