Use of synthetic circular RNA spike-ins (SynCRS) for normalization of circular RNA sequencing data
High-throughput RNA sequencing enables the quantification of transcript abundance and the identification of novel transcripts in biological samples. These include circular RNAs (circRNAs), a family of alternatively spliced RNA molecules that form a continuous loop. However, quantification and compar...
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Published in | Nature protocols |
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Main Authors | , , , |
Format | Journal Article |
Language | English |
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26.09.2024
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Abstract | High-throughput RNA sequencing enables the quantification of transcript abundance and the identification of novel transcripts in biological samples. These include circular RNAs (circRNAs), a family of alternatively spliced RNA molecules that form a continuous loop. However, quantification and comparison of circRNAs between RNA sequencing libraries remain challenging due to confounding errors introduced during exonuclease digestion, library preparation and RNA sequencing itself. Here we describe a set of synthetic circRNA spike-ins-termed 'SynCRS'-that can be added directly to purified RNA samples before exonuclease digestion and library preparation. SynCRS, introduced either individually or in combinations of varying size and abundance, can be integrated into all next-generation sequencing workflows and, critically, facilitate the quantitative calibration of circRNA transcript abundance between samples, tissue types, species and laboratories. Our step-by-step protocol details the generation of SynCRS and guides users on the stoichiometry of SynCRS spike-in to RNA samples, followed by the bioinformatic steps required to facilitate quantitative comparisons of circRNAs between libraries. The laboratory steps to produce the SynCRS require an additional 3 d on top of the high throughput circRNA sequencing and bioinformatics. The protocol is suitable for users with basic experience in molecular biology and bioinformatics. |
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AbstractList | High-throughput RNA sequencing enables the quantification of transcript abundance and the identification of novel transcripts in biological samples. These include circular RNAs (circRNAs), a family of alternatively spliced RNA molecules that form a continuous loop. However, quantification and comparison of circRNAs between RNA sequencing libraries remain challenging due to confounding errors introduced during exonuclease digestion, library preparation and RNA sequencing itself. Here we describe a set of synthetic circRNA spike-ins-termed 'SynCRS'-that can be added directly to purified RNA samples before exonuclease digestion and library preparation. SynCRS, introduced either individually or in combinations of varying size and abundance, can be integrated into all next-generation sequencing workflows and, critically, facilitate the quantitative calibration of circRNA transcript abundance between samples, tissue types, species and laboratories. Our step-by-step protocol details the generation of SynCRS and guides users on the stoichiometry of SynCRS spike-in to RNA samples, followed by the bioinformatic steps required to facilitate quantitative comparisons of circRNAs between libraries. The laboratory steps to produce the SynCRS require an additional 3 d on top of the high throughput circRNA sequencing and bioinformatics. The protocol is suitable for users with basic experience in molecular biology and bioinformatics. |
Author | Gabryelska, Marta Conn, Vanessa M Conn, Simon J Liu, Ryan |
Author_xml | – sequence: 1 givenname: Vanessa M surname: Conn fullname: Conn, Vanessa M organization: Flinders Health and Medical Research Institute, Flinders University, College of Medicine and Public Health, Bedford Park, South Australia, Australia – sequence: 2 givenname: Ryan surname: Liu fullname: Liu, Ryan organization: Flinders Health and Medical Research Institute, Flinders University, College of Medicine and Public Health, Bedford Park, South Australia, Australia – sequence: 3 givenname: Marta surname: Gabryelska fullname: Gabryelska, Marta organization: Flinders Health and Medical Research Institute, Flinders University, College of Medicine and Public Health, Bedford Park, South Australia, Australia – sequence: 4 givenname: Simon J orcidid: 0000-0002-1376-4515 surname: Conn fullname: Conn, Simon J email: simon.conn@flinders.edu.au organization: Flinders Health and Medical Research Institute, Flinders University, College of Medicine and Public Health, Bedford Park, South Australia, Australia. simon.conn@flinders.edu.au |
BackLink | https://www.ncbi.nlm.nih.gov/pubmed/39327539$$D View this record in MEDLINE/PubMed |
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References | S Roy (1053_CR12) 2022; 21 JN Vo (1053_CR9) 2019; 176 VM Conn (1053_CR17) 2023; 41 S Dodbele (1053_CR15) 2021; 22 R Drula (1053_CR1) 2024; 15 DA Cooper (1053_CR8) 2018; 1724 AF Nielsen (1053_CR13) 2022; 19 Y Li (1053_CR16) 2022; 23 T Xu (1053_CR11) 2017; 18 TB Hansen (1053_CR4) 2016; 44 PR Pandey (1053_CR20) 2019; 155 PR Pandey (1053_CR2) 2020; 11 J Salzman (1053_CR14) 2012; 7 RA Wesselhoeft (1053_CR25) 2018; 9 CJ Kershaw (1053_CR23) 2012; 941 1053_CR29 T Xu (1053_CR26) 2020; 8 SA Hardwick (1053_CR27) 2016; 13 VM Conn (1053_CR6) 2020; 9 V Conn (1053_CR7) 2019; 25 M-S Xiao (1053_CR19) 2019; 47 S Zhong (1053_CR10) 2019; 41 L Jiang (1053_CR28) 2011; 21 D Liu (1053_CR18) 2024; 52 X Zeng (1053_CR5) 2017; 13 J Zhang (1053_CR22) 2021; 39 JU Guo (1053_CR21) 2014; 15 J Zhang (1053_CR3) 2020; 11 IL Hofacker (1053_CR24) 2006; 22 |
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