During maturation, eukaryotic precursor RNAs undergo processing events including intron splicing, 3′-end cleavage, and polyadenylation. Here we describe nanopore analysis of co-transcriptional processing (nano-COP), a method for probing the timing and patterns of RNA processing. An extension of native elongating transcript sequencing, which quantifies transcription genome-wide through short-read sequencing of nascent RNA 3′ ends, nano-COP uses long-read nascent RNA sequencing to observe global patterns of RNA processing. First, nascent RNA is stringently purified through a combination of 4-thiouridine metabolic labeling and cellular fractionation. In contrast to cDNA or short-read–based approaches relying on reverse transcription or amplification, the sample is sequenced directly through nanopores to reveal the native context of nascent RNA. nano-COP identifies both active transcription sites and splice isoforms of single RNA molecules during synthesis, providing insight into patterns of intron removal and the physical coupling between transcription and splicing. The nano-COP protocol yields data within 3 d.

在成熟过程中，真核前体 RNA 会经历加工事件，包括内含子剪接、3' 端切割和聚腺苷酸化。在这里，我们描述了共转录处理 (nano-COP) 的纳米孔分析，这是一种探测 RNA 处理的时间和模式的方法。nano-COP 是原生延伸转录本测序的延伸，通过新生 RNA 3' 末端的短读长测序量化全基因组转录，nano-COP 使用长读长新生 RNA 测序来观察 RNA 加工的全局模式。首先，新生 RNA 通过 4-硫尿苷代谢标记和细胞分离相结合进行严格纯化。与依赖逆转录或扩增的 cDNA 或基于短读长的方法相比，样本直接通过纳米孔进行测序，以揭示新生 RNA 的天然环境。nano-COP 在合成过程中识别单个 RNA 分子的活性转录位点和剪接异构体，从而深入了解内含子去除模式以及转录和剪接之间的物理耦合。纳米 COP 协议在 3 d 内产生数据。