Do Trung, Qu Zhipeng, Searle Iain
Department of Molecular and Biomedical Sciences, School of Biological Sciences, The University of Adelaide, Adelaide, SA, Australia.
Methods Mol Biol. 2019;1933:131-147. doi: 10.1007/978-1-4939-9045-0_7.
More than 70% of eukaryotic genomes are transcribed into RNA transcripts, the majority of these transcripts are noncoding protein, and their biological functions are largely unknown. Over the last decade, the application of high-throughput sequencing technologies has led to the description of almost all cellular coding and noncoding RNA transcripts except perhaps for those transcripts that are lowly abundant or those present only in specific cells that are underrepresented in sampled tissue(s). An often underrepresented class of noncoding are long noncoding RNAs (lncRNAs), and these often play key regulatory functions for many biological processes such as cell identity and cell division. However, the purification and functional characterization in vitro are still a challenge in both animal and plant experimental systems. Here, we describe in detail methodology for purification of specific cell types, bioinformatic annotation of lncRNAs, and investigation of biological function using the reference plant Arabidopsis thaliana.
超过70%的真核生物基因组被转录为RNA转录本，其中大多数转录本是非编码蛋白质的，其生物学功能大多未知。在过去十年中，高通量测序技术的应用使得几乎所有细胞编码和非编码RNA转录本都得以描述，可能除了那些低丰度的转录本或仅存在于取样组织中代表性不足的特定细胞中的转录本。一类经常代表性不足的非编码RNA是长链非编码RNA（lncRNA），它们通常在许多生物学过程中发挥关键调节功能，如细胞身份和细胞分裂。然而，在动物和植物实验系统中，lncRNA的体外纯化和功能表征仍然是一个挑战。在这里，我们详细描述了使用参考植物拟南芥纯化特定细胞类型的方法、lncRNA的生物信息学注释以及生物学功能研究。
