He Feng, Celik Alper, Baker Richard, Jacobson Allan
Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, MA, United States.
Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, MA, United States.
Methods Enzymol. 2018;612:147-181. doi: 10.1016/bs.mie.2018.09.005. Epub 2018 Oct 9.
Contemporary high-throughput sequencing methods, notably RNA-Seq, permit the systematic identification and characterization of transcripts whose levels change significantly in response to altered biological states. We have described methods for the application of this methodology to a definition of the transcripts regulated by the NMD pathway in the yeast Saccharomyces cerevisiae. In short, we outline methods for growing cells of wild-type or NMD-deficient yeast, isolating RNA from the different strains, depleting rRNA from each sample, preparing and sequencing the respective RNA-Seq libraries, and employing multiple software packages to characterize the resulting sequence reads meaningfully. Our experimental approach has identified approximately 900 transcripts that are commonly upregulated when yeast NMD is inactivated (Celik, Baker, He, & Jacobson, 2017).
当代高通量测序方法，尤其是RNA测序，能够系统地鉴定和表征那些因生物状态改变而水平发生显著变化的转录本。我们已经描述了将该方法应用于定义酿酒酵母中受NMD途径调控的转录本的方法。简而言之，我们概述了培养野生型或NMD缺陷型酵母细胞、从不同菌株中分离RNA、从每个样本中去除rRNA、制备并测序各自的RNA测序文库，以及使用多个软件包对所得序列读数进行有意义表征的方法。我们的实验方法已经鉴定出约900个转录本，当酵母NMD失活时，这些转录本通常会上调（Celik、Baker、He和Jacobson，2017年）。
