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‘Read–through marking’ reveals differential nucleotide composition of read-through and truncated cDNAs in iCLIP

We established a modified iCLIP protocol, called ‘read-through marking’, which facilitates the detection of cDNAs that have not been truncated upon encountering the RNA–peptide complex during reverse transcription (read-through cDNAs). A large proportion of these cDNAs would be undesirable in an iCL...

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Detalles Bibliográficos
Autores principales: Huppertz, Ina, Haberman, Nejc, Ule, Jernej
Formato: Online Artículo Texto
Lenguaje:English
Publicado: F1000 Research Limited 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6051193/
https://www.ncbi.nlm.nih.gov/pubmed/30057947
http://dx.doi.org/10.12688/wellcomeopenres.14663.1
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author Huppertz, Ina
Haberman, Nejc
Ule, Jernej
author_facet Huppertz, Ina
Haberman, Nejc
Ule, Jernej
author_sort Huppertz, Ina
collection PubMed
description We established a modified iCLIP protocol, called ‘read-through marking’, which facilitates the detection of cDNAs that have not been truncated upon encountering the RNA–peptide complex during reverse transcription (read-through cDNAs). A large proportion of these cDNAs would be undesirable in an iCLIP library, as it could affect the resolution of the method. To this end, we added an oligonucleotide to the 5’-end of RNA fragments—a 5’-marker—to mark the read-through cDNAs. By applying this modified iCLIP protocol to PTBP1 and eIF4A3, we found that the start sites of read-through cDNAs are enriched in adenosines, while the remaining cDNAs have a markedly different sequence content at their starts, preferentially containing thymidines. This finding in turn indicates that most of the reads in our iCLIP libraries are a product of truncation with valuable information regarding the proteins’ RNA-binding sites. Thus, cDNA start sites confidently identify a protein’s RNA-crosslink sites and we can account for the impact of read-through cDNAs by commonly adding a 5’-marker.
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spelling pubmed-60511932018-07-27 ‘Read–through marking’ reveals differential nucleotide composition of read-through and truncated cDNAs in iCLIP Huppertz, Ina Haberman, Nejc Ule, Jernej Wellcome Open Res Research Note We established a modified iCLIP protocol, called ‘read-through marking’, which facilitates the detection of cDNAs that have not been truncated upon encountering the RNA–peptide complex during reverse transcription (read-through cDNAs). A large proportion of these cDNAs would be undesirable in an iCLIP library, as it could affect the resolution of the method. To this end, we added an oligonucleotide to the 5’-end of RNA fragments—a 5’-marker—to mark the read-through cDNAs. By applying this modified iCLIP protocol to PTBP1 and eIF4A3, we found that the start sites of read-through cDNAs are enriched in adenosines, while the remaining cDNAs have a markedly different sequence content at their starts, preferentially containing thymidines. This finding in turn indicates that most of the reads in our iCLIP libraries are a product of truncation with valuable information regarding the proteins’ RNA-binding sites. Thus, cDNA start sites confidently identify a protein’s RNA-crosslink sites and we can account for the impact of read-through cDNAs by commonly adding a 5’-marker. F1000 Research Limited 2018-06-22 /pmc/articles/PMC6051193/ /pubmed/30057947 http://dx.doi.org/10.12688/wellcomeopenres.14663.1 Text en Copyright: © 2018 Huppertz I et al. http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Note
Huppertz, Ina
Haberman, Nejc
Ule, Jernej
‘Read–through marking’ reveals differential nucleotide composition of read-through and truncated cDNAs in iCLIP
title ‘Read–through marking’ reveals differential nucleotide composition of read-through and truncated cDNAs in iCLIP
title_full ‘Read–through marking’ reveals differential nucleotide composition of read-through and truncated cDNAs in iCLIP
title_fullStr ‘Read–through marking’ reveals differential nucleotide composition of read-through and truncated cDNAs in iCLIP
title_full_unstemmed ‘Read–through marking’ reveals differential nucleotide composition of read-through and truncated cDNAs in iCLIP
title_short ‘Read–through marking’ reveals differential nucleotide composition of read-through and truncated cDNAs in iCLIP
title_sort ‘read–through marking’ reveals differential nucleotide composition of read-through and truncated cdnas in iclip
topic Research Note
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6051193/
https://www.ncbi.nlm.nih.gov/pubmed/30057947
http://dx.doi.org/10.12688/wellcomeopenres.14663.1
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