Generation of full-length circRNA libraries for Oxford Nanopore long-read sequencing v2
Authors: Fuchs Steffen 
Published: September 2023

Circular RNA (circRNA) is a noncoding RNA class with broad implications for gene expression regulation, mostly by e.g. interaction with other RNAs or RNA-binding proteins. However, their specific sequence is not revealed by the commonly applied short-read Illumina sequencing. Here, we present an adapted protocol to enrich and sequence full-length circRNAs using the Oxford Nanopore long-read sequencing platform. The protocol involves an RNaseH-based ribodepletion, 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 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. The recommended amount of starting material is 7 µg of RNA, however lower amounts of RNA have also been tested successfully. Multiplexing of up to 4 libraries yields in total more than 1-2 Mio 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 Nanopore sequencing, the bioinformatic analysis of the created data, as well as the limitations of our approach and recommendations for troubleshooting and results interpretation. Taken together, this protocol enables reliable full-length analysis of circRNAs, a non-coding RNA type involved in a growing number of physiologic and pathologic conditions. Steps Enrichment of circRNAs for generation of Nanopore sequencing libraries 1. Ribodepletion 2. circRNA enrichment 3. cDNA library creation 4. Quality control Suggestions for Nanopore sequencing and data analysis 5. Suggestions for Nanopore sequencing 6. Recommendations for bioinformatics analysis of the data Expected results, limitations and troubleshooting 7. Expected results and interpretation 8. Limitations and challenges 9. Troubleshooting The enrichment of the whole circRNA fraction follows the published workflow from Zhang et al. with several modifications: Modification of the ribodepletion method from a commercial kit to the published method of Baldwin et al., which is based on a pool of DNA oligonucleotides that hybridize with ribosomal RNA and a digest of DNA:RNA hybrids by RNaseH Clean-up steps and final size selection have been adapted to select for circRNAs longer than 200 nt and therefore also consider shorter circRNAs, whereas in the original protocol a selection of > 1kb was applied (the average length of circRNAs is between 200-800 nt) Additional negative poly(A) selection for further enrichment of circRNAs Increased quantity of retrieved library by using more starting material and a higher number of PCR cycles Thorough quality control by combining qRT-PCR, Qubit and Fragment Analyzer after circRNA enrichment Further, we refer to the used sequencing protocol for the Nanopore platform, suggest changes to the standard protocol and further give recommendations for the bioinformatics analysis. We pool 4 libraries. The enrichment workflow can therefore be performed in parallel for 4 samples. (The last step in this version contains a supplemental video with extra context and tips, as part of the protocols.io Spotlight series, featuring conversations with protocol authors.)