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Immunoprecipitation and High–Throughput Sequencing of ARGONAUTE–Bound Target RNAs from Plants

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Immunoprecipitation and High–Throughput Sequencing of ARGONAUTE–Bound Target RNAs from Plants

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dc.contributor.author Carbonell Olivares, Alberto es_ES
dc.contributor.editor Carbonell Olivares, Alberto es_ES
dc.date.accessioned 2018-05-22T09:03:36Z
dc.date.available 2018-05-22T09:03:36Z
dc.date.issued 2017-06-13
dc.identifier.isbn 978-1-4939-7165-7
dc.identifier.isbn 978-1-4939-7164-0
dc.identifier.issn 1064-3745
dc.identifier.uri http://hdl.handle.net/10251/102403
dc.description.abstract ARGONAUTE (AGO) proteins function in small RNA (sRNA)-based RNA silencing pathways to regulate gene expression and control invading nucleic acids. In posttranscriptional RNA silencing pathways, plant AGOs associate with sRNAs to interact with highly sequence-complementary target RNAs. Once the AGO–sRNA-target RNA ternary complex is formed, target RNA is typically repressed through AGO-mediated cleavage or through other cleavage-independent mechanisms. The universe of sRNAs associating with diverse plant AGOs has been determined though AGO immunoprecipitation (IP) and high-throughput sequencing of co-immunoprecipitated sRNAs. To better understand the biological functions of AGO–sRNA complexes, it is crucial to identify the repertoire of target RNAs they regulate. Here I present a detailed AGO–RNA IP followed by high-throughput sequencing (AGO RIP-Seq) methodology for the isolation of AGO ternary complexes from plant tissues and the high-throughput sequencing of AGO-bound target RNAs. In particular, the protocol describes the IP of slicer-deficient hemagglutinin (HA)-tagged AGO proteins expressed in plant tissues, the isolation of AGO-bound RNAs, and the generation of target RNA libraries for high-throughput sequencing. es_ES
dc.description.sponsorship This work was supported by an Individual Fellowship from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No. 655841 to A.C. es_ES
dc.language Inglés es_ES
dc.publisher Springer Link es_ES
dc.relation.ispartof Plant Argonaute Proteins: Methods and Protocols es_ES
dc.rights Reserva de todos los derechos es_ES
dc.subject ARGONAUTE es_ES
dc.subject RNA immunoprecipitation es_ES
dc.subject High-throughput sequencing n es_ES
dc.subject RIP-Seq es_ES
dc.subject Small RNA es_ES
dc.subject RNA-Seq es_ES
dc.subject Library preparatio es_ES
dc.title Immunoprecipitation and High–Throughput Sequencing of ARGONAUTE–Bound Target RNAs from Plants es_ES
dc.type Capítulo de libro es_ES
dc.identifier.doi 10.1007/978-1-4939-7165-7_6
dc.relation.projectID info:eu-repo/grantAgreement/EC/H2020/655841/EU/Genome-wide analysis of RNA and protein interacting profiles during a plant virus infection/ es_ES
dc.rights.accessRights Abierto es_ES
dc.contributor.affiliation Universitat Politècnica de València. Instituto Universitario Mixto de Biología Molecular y Celular de Plantas - Institut Universitari Mixt de Biologia Molecular i Cel·lular de Plantes es_ES
dc.description.bibliographicCitation Carbonell Olivares, A. (2017). Immunoprecipitation and High–Throughput Sequencing of ARGONAUTE–Bound Target RNAs from Plants. En Plant Argonaute Proteins: Methods and Protocols. Springer Link. 93-112. https://doi.org/10.1007/978-1-4939-7165-7_6 es_ES
dc.relation.publisherversion http://dx.doi.org/10.1007/978-1-4939-7165-7_6 es_ES
dc.description.upvformatpinicio 93 es_ES
dc.description.upvformatpfin 112 es_ES
dc.identifier.eissn 1940-6029
dc.contributor.funder European Commission es_ES
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