Generation of full-length circRNA libraries for Oxford Nanopore long-read sequencing v1

Abstract

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 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. 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. 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 Ribodepletion circRNA enrichment cDNA library creation Quality control The enrichment of the whole circRNA fraction follows the published workflow from Zhang et al. with several modifications: Modification of the ribodepletion method 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 reference the used sequencing protocol for the Nanopore platform and provide suggestions for the bioinformatics analysis.