Department of Developmental Biology, Sloan-Kettering Institute, New York, NY 10065, USA; Louis V. Gerstner, Jr. Graduate School of Biomedical Sciences, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA.
Department of Biology, University of Nevada, Reno, Reno, NV 89557, USA.
Methods. 2017 Aug 15;126:86-94. doi: 10.1016/j.ymeth.2017.06.003. Epub 2017 Jun 8.
Alternative polyadenylation (APA) diversifies the 3' termini of a majority of mRNAs in most eukaryotes, and is consequently inferred to have substantial consequences for the utilization of post-transcriptional regulatory mechanisms. Since conventional RNA-sequencing methods do not accurately define mRNA termini, a number of protocols have been developed that permit sequencing of the 3' ends of polyadenylated transcripts (3'-seq). We present here our experimental protocol to generate 3'-seq libraries using a dT-priming approach, including extensive details on considerations that will enable successful library cloning. We pair this with a set of computational tools that allow the user to process the raw sequence data into a filtered set of clusters that represent high-confidence functional polyadenylation sites. The data are single-nucleotide resolution and quantitative, and can be used for downstream analyses of APA.
可变多聚腺苷酸化 (APA) 使大多数真核生物的大多数 mRNA 的 3' 末端多样化，因此被认为对转录后调控机制的利用有重大影响。由于传统的 RNA 测序方法不能准确地定义 mRNA 末端，因此开发了许多允许对多聚腺苷酸化转录本的 3' 末端进行测序的方案 (3'-seq)。我们在此介绍了一种使用 dT-引物方法生成 3'-seq 文库的实验方案，其中包括关于可实现文库克隆成功的考虑因素的详细信息。我们将其与一组计算工具结合使用，这些工具允许用户将原始序列数据处理为一组过滤后的簇，这些簇代表高可信度的功能多聚腺苷酸化位点。该数据为单核苷酸分辨率且定量，可以用于 APA 的下游分析。
