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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.

Simple Summary Anaplastic lymphoma kinase (ALK) is a tyrosine kinase associated with Anaplastic Large Cell lymphoma (ALCL) through oncogenic translocations mainly NPM-ALK. Chemotherapy is effective in ALK(+) ALCL patients and induces remission rates of approximately 80%. The remaining patients do not respond to chemotherapy and some patients have drug-resistant relapses. Different classes of ALK tyrosine kinase inhibitors (TKI) are available but used exclusively for EML4-ALK (+) lung cancers. The significant toxicities of most ALK inhibitors explain the delay in their use in pediatric ALCL patients. Some ALCL patients do not respond to the first generation TKI or develop an acquired resistance. Combination therapy with ALK inhibitors in ALCL is the current challenge. Abstract Initially discovered in anaplastic large cell lymphoma (ALCL), the ALK anaplastic lymphoma kinase is a tyrosine kinase which is affected in lymphomas by oncogenic translocations, mainly NPM-ALK. To date, chemotherapy remains a viable option in ALCL patients with ALK translocations as it leads to remission rates of approximately 80%. However, the remaining patients do not respond to chemotherapy and some patients have drug-resistant relapses. It is therefore crucial to identify new and better treatment options. Nowadays, different classes of ALK tyrosine kinase inhibitors (TKI) are available and used exclusively for EML4-ALK (+) lung cancers. In fact, the significant toxicities of most ALK inhibitors explain the delay in their use in ALCL patients, who are predominantly children. Moreover, some ALCL patients do not respond to Crizotinib, the first generation TKI, or develop an acquired resistance months following an initial response. Combination therapy with ALK inhibitors in ALCL is the current challenge.