| Low-cost and High-throughput RNA-seq Library Preparation for Illumina Sequencing from Plant Tissue
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Author:
Date:
2020-10-20
[Abstract] 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 ...
[摘要] [摘要] 转录组分析可以为不同遗传背景或不同条件下的生物学过程提供线索。RNA测序（RNA-seq）的价格已经下降到足够低的程度，因此在各种条件下进行中大规模转录组分析是可行的。然而，对于那些希望第一次使用RNA-seq或进行单个实验的人来说，RNA-seq库制备的价格和各种选择仍然是令人望而生畏的。选择文库制备方案的标准包括RNA分离方法、核苷酸片段化以获得所需的大小范围，以及文库索引以汇集测序样本进行多路复用。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mRNA制备文库用于转录组分析。高质量和高通量的方案选择都包括通过磁珠使poly-A尾部沉淀，cDNA合成，然后通过Tn5介导的“标记”同时裂解和添加适配器的步骤。该方案的所有步骤均已通过拟南芥叶片和幼苗组织的验证，并简化为协同工作，在资金和时间上成本最低，因此旨在为转录组分析提供一个初学者友好的从开始到完成的RNA序列库制备。
[背景] 通过Southern印迹、expressed sequence ...
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| Preparation of cDNA Library for dRNA-seq
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Author:
Date:
2012-12-05
[Abstract] microRNAs (miRNAs) are ubiquitous regulators of gene expression in eukaryotic organisms, which guide Argonaute proteins (AGO) to cleave target mRNA or inhibit its translation based on sequence complementarity. In plants, miRNA directed cleavage occurs on the target mRNA at about 10 to 11 nucleotide (nt) up stream to the site where the 5’ end of miRNA binds. Sequencing of the miRNA directed cleavage products (degradome) is widely employed as a way to both verify bioinformatic predictions of miRNA mediated regulation and identify novel targets of known miRNAs. Here we describe a protocol for preparation of degradome RNA complementary DNA library for high-through-put sequencing (dRNA-seq) using Illumina GA II sequencing platform, which is currently most popular and cost-effective. Using this ...
[摘要] 微小RNA（miRNA）是真核生物体中基因表达的普遍存在的调节剂，其指导Argonaute蛋白（AGO）基于序列互补性切割靶mRNA或抑制其翻译。在植物中，miRNA定向切割发生在靶mRNA上，在约5至11个核苷酸（nt）上游至miRNA的5'端结合的位点。 miRNA定向切割产物（降解组）的测序广泛用作验证miRNA介导的调节的生物信息学预测和鉴定已知miRNA的新靶标的方法。在这里，我们描述了使用Illumina GA II测序平台制备高通量测序（dRNA-seq）的降解组RNA互补DNA文库的方案，目前这是最受欢迎和成本效益的。使用这个协议，我们成功地使用三个茄科植物，包括烟草，番茄和马铃薯生成三个dRNA seq图书馆。尽管该协议是用单链适配器开发的，但应该能够通过用多路复用兼容的3'衔接子代替3'衔接子并用索引引物代替PCR引物来产生多重文库。
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