Elongation, splicing and polyadenylation are fundamental steps of transcription, and studying their coordination requires simultaneous monitoring of these dynamic processes on one transcript. We recently developed a full-length nascent RNA sequencing method in the model plant Arabidopsis that simultaneously detects RNA polymerase II position, splicing status, polyadenylation site and poly(A) tail length at genome-wide scale. This method allows calculation of the kinetics of cotranscriptional splicing and detects polyadenylated transcripts with unspliced introns retained at specific positions posttranscriptionally. Here we describe a detailed protocol for this method called FLEP-seq (full-length elongating and polyadenylated RNA sequencing) that is applicable to plants. Library production requires as little as one nanogram of nascent RNA (after rRNA/tRNA removal), and either Nanopore or PacBio platforms can be used for sequencing. We also provide a complete bioinformatic pipeline from raw data processing to downstream analysis. The minimum time required for FLEP-seq, including RNA extraction and library preparation, is 36 h. The subsequent long-read sequencing and initial data analysis ranges between 31 and 40 h, depending on the sequencing platform.

延伸、剪接和多聚腺苷酸化是转录的基本步骤，研究它们的协调需要同时监测一个转录本上的这些动态过程。我们最近在模式植物拟南芥中开发了一种全长新生 RNA 测序方法在全基因组范围内同时检测 RNA 聚合酶 II 的位置、剪接状态、多聚腺苷酸化位点和多聚 (A) 尾长。该方法允许计算共转录剪接的动力学，并检测转录后保留在特定位置的未剪接内含子的多腺苷酸化转录本。在这里，我们描述了这种方法的详细协议，称为 FLEP-seq（全长延伸和多腺苷酸化 RNA 测序），适用于植物。文库生产只需 1 纳克新生 RNA（去除 rRNA/tRNA 后），Nanopore 或 PacBio 平台均可用于测序。我们还提供从原始数据处理到下游分析的完整生物信息学管道。FLEP-seq 所需的最短时间（包括 RNA 提取和文库制备）为 36 小时。