Enrichment and analysis of circular RNA by Induro RT-mediated circRNA sequencing (IMCR-seq)
Circular RNA (circRNA) has recently gained attention for its emerging biological activities, relevance to disease, and potential as biomarkers. Furthermore, the growing prominence of RNA vaccines has brought circRNAs into the spotlight as a promising alternative modality due to their enhanced stability compared to linear RNA. Nevertheless, sequencing circRNAs has presented challenges. In this context, we introduce a novel circRNA sequencing method called Induro-RT mediated circRNA-sequencing (IMCR-seq), which relies on a group II intron reverse transcriptase with robust rolling circle reverse transcription activity. The IMCR-seq protocol eliminates the need for conventional circRNA enrichment methods such as rRNA depletion and RNaseR digestion yet achieved the highest circRNA enrichment and detected 6-1000 times more circRNAs for the benchmarked human samples compared to other methods. IMCR-seq is applicable to any organism, capable of detecting circRNAs of more than 7,000 nucleotides, and is effective using as little as 10 ng of total RNA. These enhancements render IMCR-seq suitable for clinical samples, including disease tissues and liquid biopsies. To demonstrate IMCR-seq's clinical relevance, we applied it to lung tumor tissue and plasma samples from a healthy individual and a lung cancer patient and detected cancer-specific circRNAs as potential biomarkers. In summary, IMCR-seq presents an efficient and versatile circRNA sequencing method with high potential for research and clinical applications. Overall design: We developed a new circRNA sequencing method called Induro-RT mediated circRNA-sequencing (IMCR-seq) by using the group II intron reverse transcriptase Induro-RT and long read sequencing technologies. We applied IMCR-seq to profile circRNAs of human HEK293 cell line, human brain and human lung tumor tissues, and plasma samples from a healthy individual and a lung cancer patient