High-throughput sequencing of small RNAs in Nicotiana tabacum
Ontology highlight
ABSTRACT:
ORGANISM(S): Nicotiana tabacum
PROVIDER: GSE28977 | GEO | 2011/05/10
SECONDARY ACCESSION(S): PRJNA142831
REPOSITORIES: GEO
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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 Nicotiana tabacum tissues (including leaves, flowers and pods). 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 maize genome under study. Small RNA libraries were derived from leaves, flowers and pods of Nicotiana tabacum. Total RNA was isolated using the TriReagent (Molecular Research Center) for leaves and flowers, and the Plant RNA Purification Reagent (Invitrogen) for pods, 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 Barbara Baker for providing the plant material as well as Kan Nobuta and Gayathri Mahalingam for assistance with the computational methods.
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