Direct RNA Sequencing with Nanopore technology is a cutting-edge method for sequencing RNA molecules without the need for conversion to complementary DNA (cDNA) as required in traditional RNA sequencing methods. The protocol involves the direct sequencing of RNA strands through nanopores, allowing for the real-time detection of RNA sequences and RNA bases modifications. Direct RNA Sequencing with Nanopore technology offers the advantage of studying RNA molecules in their native state, providing valuable insights into RNA processing, alternative splicing, and modifications without the need for reverse transcription. It is particularly valuable for capturing the full complexity of the transcriptome.