Low-cost and High-throughput RNA-seq Library Preparation for Illumina Sequencing from Plant Tissue
Low-cost and High-throughput RNA-seq Library Preparation for Illumina Sequencing from Plant Tissue
复制标题
DOI：
10.21769/bioprotoc.3799
复制
发表时间：
2020-10-20
期刊：
影响因子：
0.8
通讯作者：
Ding, Pingtao
中科院分区：
文献类型：
--
作者：
Bjornson, Marta;Kajala, Kaisa;Ding, Pingtao
转录组分析可以为不同遗传背景或/和各种条件下受影响的生物过程提供线索。RNA测序（RNA-seq）的价格已经足够低，因此在一系列条件下进行中到大规模的转录组分析是可行的。然而，RNA-seq文库制备的价格和选择的多样性对于那些希望首次使用RNA-seq或进行单一实验的人来说仍然是令人生畏的。选择文库制备方案的标准包括RNA分离的方法、获得所需大小范围的核苷酸片段化以及文库索引以汇集测序样品用于多重化。在这里，我们提出了一个高质量和高通量的选择，从多聚腺苷酸化mRNA的转录组分析准备库。高质量和高通量方案选项均包括通过磁珠使能的poly-A尾沉淀的mRNA富集步骤、cDNA合成，然后通过Tn 5介导的“标签化”同时进行片段化和衔接子添加。该方案的所有步骤都已经用拟南芥叶和幼苗组织进行了验证，并以最小的金钱和时间成本简化为一起工作，因此旨在为转录组分析提供初学者友好的开始到结束的RNA-seq文库制备。
Transcriptome analysis can provide clues to biological processes affected in different genetic backgrounds or/and under various conditions. The price of RNA sequencing (RNA-seq) has decreased enough so that medium- to large-scale transcriptome analyses in a range of conditions are feasible. However, the price and variety of options for library preparation of RNA-seq can still be daunting to those who would like to use RNA-seq for their first time or for a single experiment. Among the criteria for selecting a library preparation protocol are the method of RNA isolation, nucleotide fragmentation to obtain desired size range, and library indexing to pool sequencing samples for multiplexing. Here, we present a high-quality and a high-throughput option for preparing libraries from polyadenylated mRNA for transcriptome analysis. Both high-quality and high-throughput protocol options include steps of mRNA enrichment through magnetic bead-enabled precipitation of the poly-A tail, cDNA synthesis, and then fragmentation and adapter addition simultaneously through Tn5-mediated 'tagmentation'. All steps of the protocols have been validated with Arabidopsis thaliana leaf and seedling tissues and streamlined to work together, with minimal cost in money and time, thus intended to provide a beginner-friendly start-to-finish RNA-seq library preparation for transcriptome analysis.