NAP-seq for full-length noncapped RNA sequencing.

The majority of the mammalian genome is transcribed into RNAs, most of which are noncapped RNAs (napRNAs) that not only regulate diverse biological processes through their functions as noncoding RNAs but also serve as processing products to delineate specific RNA biogenesis pathways. However, due to their heterogeneous lengths, diverse terminal modifications and complex secondary structures, identifying these napRNAs poses substantial challenges. Recently, we developed a napRNA sequencing technique (NAP-seq) to identify full-length sequences of napRNAs with various terminal modifications at single-nucleotide resolution. Here we describe the experimental design principles and detailed step-by-step procedures for discovering napRNAs across multiple cell types. The procedure includes T4 polynucleotide kinase pretreatment to standardize RNA termini, enabling comprehensive capture of modified napRNAs; size-selection followed by depletion of known high-abundance RNAs via RNase H to enrich long and low-abundance RNAs; and use of custom-designed adapters with random barcodes, permitting identification of full-length napRNAs at single-nucleotide resolution while minimizing PCR biases and adapter ligation inefficiencies. The use of thermally stable reverse transcriptase enzymes and nested reverse transcriptase primers ensures full-length cDNA synthesis across structured or modified RNA regions while minimizing mispriming artifacts. Libraries are sequenced in parallel using Oxford Nanopore (long-read) and Illumina (short-read) platforms, synergizing advantages of third-generation and next-generation sequencing technologies. The entire experimental procedure, from library preparation to deep sequencing and computational analysis, can be completed within 8 d. The NAP-seq approach enables researchers to discover novel classes of noncoding RNAs with regulatory functions and to investigate RNA biogenesis in various tissues and cell lines.