Nuclear-m⁶A-Label-Seq Enables Transcriptome-Wide Nuclear m⁶A Profiling at Single-Base Resolution

N⁶-Methyladenosine (m⁶A) on mature mRNA has been extensively characterized, yet its precise mapping and functions in nuclear noncoding RNAs remain elusive. To address this issue, we recently developed Nuclear-m⁶A-label-seq, a metabolic labeling-based method for transcriptome-wide nuclear m⁶A profiling at single-base resolution. This approach builds on the prototypical m⁶A-label-seq principle, in which an allyl group, instead of methyl group, is metabolically installed at N⁶-position at supposed RNA m⁶A-generating adenosines and the resultant N⁶-allyl adenosine is subsequently converted into 1, N⁶-cyclized adenosine (cyc-A) by mild iodination reaction. During RNA reverse transcription, HIV reverse transcriptase is employed to introduce a base misincorporation at cyc-A sites while enabling a template switch to incorporate adapter sequences to the complementary DNA end in a single step. Through this strategy, library construction is shortened to about 6 h, and the required cell-labeling total RNA input is reduced to 5 μg of total nuclear RNA, representing a 100-fold reduction compared to the prototypical protocol. Both polyadenylated and nonpolyadenylated nuclear transcripts are captured through the sequential nuclear RNA isolation and rRNA depletion. Following high-throughput sequencing, reads from human cells are aligned with the complete T2T-CHM13 genome, enabling accurate mapping of repetitive regions. Aligned reads are then analyzed using the user-friendly rMATS-DVR pipeline to identify high-confidence m⁶A sites based on cyc-A-induced misincorporation patterns. Here, we provide a detailed step-by-step protocol for Nuclear-m⁶A-label-seq, which stands for a direct and high-resolution approach for profiling the nuclear m⁶A epitranscriptome.