dc.contributor.author
Fuchs, Steffen
dc.contributor.author
Babin, Loélia
dc.contributor.author
Andraos, Elissa
dc.contributor.author
Bessiere, Chloé
dc.contributor.author
Willier, Semjon
dc.contributor.author
Schulte, Johannes H.
dc.contributor.author
Gaspin, Christine
dc.contributor.author
Meggetto, Fabienne
dc.date.accessioned
2023-04-27T13:59:21Z
dc.date.available
2023-04-27T13:59:21Z
dc.identifier.uri
https://refubium.fu-berlin.de/handle/fub188/39147
dc.identifier.uri
http://dx.doi.org/10.17169/refubium-38864
dc.description.abstract
Circular RNA (circRNA) is a noncoding RNA class with important implications for gene expression regulation, mostly by interaction with other RNA species or RNA-binding proteins. While the commonly applied short-read Illumina RNA-sequencing techniques can be used to detect circRNAs, their full sequence is not revealed. However, the complete sequence information is needed to analyze potential interactions and thus the mechanism of action of circRNAs. Here, we present an improved protocol to enrich and sequence full-length circRNAs by using the Oxford Nanopore long-read sequencing platform. The protocol involves an enrichment of lowly abundant circRNAs by exonuclease treatment and negative selection of linear RNAs. Then, a cDNA library is created and amplified by PCR. This protocol provides enough material for several sequencing runs. The library is used as input for ligation-based sequencing together with native barcoding. Stringent quality control of the libraries is ensured by a combination of Qubit, Fragment Analyzer and qRT-PCR. Multiplexing of up to 4 libraries yields in total more than 1-2 Million reads per library, of which 1-2% are circRNA-specific reads with > 99% of them full-length. The protocol works well with human cancer cell lines. We further provide suggestions for the bioinformatic analysis of the created data, as well as the limitations of our approach together with recommendations for troubleshooting and interpretation. Taken together, this protocol enables reliable full-length analysis of circRNAs, a noncoding RNA type involved in a growing number of physiologic and pathologic conditions.
en
dc.rights.uri
https://creativecommons.org/licenses/by/4.0/
dc.subject
Oxford Nanopore long-read sequencing
en
dc.subject
full-length circular RNA libraries
en
dc.subject
gene expression regulation
en
dc.subject.ddc
600 Technik, Medizin, angewandte Wissenschaften::610 Medizin und Gesundheit::610 Medizin und Gesundheit
dc.title
Generation of full-length circular RNA libraries for Oxford Nanopore long-read sequencing
dc.type
Wissenschaftlicher Artikel
dcterms.bibliographicCitation.articlenumber
e0273253
dcterms.bibliographicCitation.doi
10.1371/journal.pone.0273253
dcterms.bibliographicCitation.journaltitle
PLOS ONE
dcterms.bibliographicCitation.number
9
dcterms.bibliographicCitation.originalpublishername
Public Library of Science (PLoS)
dcterms.bibliographicCitation.volume
17
refubium.affiliation
Charité - Universitätsmedizin Berlin
refubium.resourceType.isindependentpub
no
dcterms.accessRights.openaire
open access
dcterms.bibliographicCitation.pmid
36070299
dcterms.isPartOf.eissn
1932-6203