pNAD-seq: a highly sensitive and accurate method for profiling of NAD-capped transcriptomes

The discovery of NAD+ cap in RNAs suggests a novel gene regulation mechanism. Existing NAD-capped RNA (NAD-RNA) detecting methods lack precision in determining 5' ends or covering smaller NAD-RNAs. Our precision NAD-RNA sequencing (pNAD-seq) utilizes a dual enrichment approach for accurate 5' end determination and comprehensive coverage of shorter NAD-RNAs. We also developed NAD-linkSeq for full-length NAD-RNA sequencing. These methods revealed a broader array of NAD-RNAs in E. coli. Notably, NAD-RNAs from protein-coding genes are small-medium, originated from various gene regions or as antisense transcripts, suggesting regulatory functions. This expanded NAD-RNA repertoire offers insights into NAD capping mechanisms and functions.