RACE-Nano-Seq: Profiling Transcriptome Diversity of a Genomic Locus.

The complexity of the human transcriptome poses significant challenges for complete annotation. Traditional RNA-seq, often limited by sensitivity and short read lengths, is frequently inadequate for identifying low-abundant transcripts and resolving complex populations of transcript isoforms. Direct long-read sequencing, while offering full-length information, suffers from throughput limitations, hindering the capture of low-abundance transcripts. To address these challenges, we introduce a targeted RNA enrichment strategy, rapid amplification of cDNA ends coupled with Nanopore sequencing (RACE-Nano-Seq). This method unravels the deep complexity of transcripts containing anchor sequences-specific regions of interest that might be exons of annotated genes, in silico predicted exons, or other sequences. RACE-Nano-Seq is based on inverse PCR with primers targeting these anchor regions to enrich the corresponding transcripts in both 5' and 3' directions. This method can be scaled for high-throughput transcriptome profiling by using multiplexing strategies. Through targeted RNA enrichment and full-length sequencing, RACE-Nano-Seq enables accurate and comprehensive profiling of low-abundance transcripts, often revealing complex transcript profiles at the targeted loci, both annotated and unannotated. Key features • This protocol is highly sensitive and can detect low-abundance transcripts. • This protocol can be performed in a typical molecular biology laboratory. • This protocol allows RACE reactions with single or multiple primers, supporting various research scales. • This protocol enables characterization of complex genomic loci and discovery of novel transcripts, exons, and alternative splicing events.