| Low-cost and High-throughput RNA-seq Library Preparation for Illumina Sequencing from Plant Tissue
|
|
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 ...
|
|
|
| A Method for User-defined Mutagenesis by Integrating Oligo Pool Synthesis Technology with Nicking Mutagenesis
|
|
Author:
Date:
2020-08-05
[Abstract] Saturation mutagenesis is a fundamental enabling technology for protein engineering and epitope mapping. Nicking mutagenesis (NM) allows the user to rapidly construct libraries of all possible single mutations in a target protein sequence from plasmid DNA in a one-pot procedure. Briefly, one strand of the plasmid DNA is degraded using a nicking restriction endonuclease and exonuclease treatment. Mutagenic primers encoding the desired mutations are annealed to the resulting circular single-stranded DNA, extended with high-fidelity polymerase, and ligated into covalently closed circular DNA by Taq DNA ligase. The heteroduplex DNA is resolved by selective degradation of the template strand. The complementary strand is synthesized and ligated, resulting in a library of mutated ...
[摘要] [摘要] 饱和突变是蛋白质工程和表位定位的基础技术。缺口突变（NM）允许用户在一锅程序中快速构建目标蛋白序列中所有可能的单一突变的文库。简言之，质粒DNA的一条链被切痕限制性内切酶和核酸外切酶处理降解。编码所需突变的突变引物退火成环状单链DNA，用高保真聚合酶延伸，并通过taqdna连接酶连接成共价闭合的环状DNA。通过选择性降解模板链来分解异源双链DNA。合成并连接互补链，形成一个共价闭合的环状质粒库。后来的研究表明，由于该过程中使用的引物很少，因此通常在使用前需要扩增的再悬浮寡聚物池可以直接用于诱变过程。因为寡聚体可以包含数以万计的独特寡聚体，这使得在一个单罐突变反应中能够构建数以万计用户定义突变的库，这显著提高了NM的效用，如下所述。
寡聚物的使用为诱变实验提供了一种经济上有利的方法。首先，寡核苷酸池合成比传统合成便宜得多。第二，可以产生一个混合池，用于多个不同基因的诱变。为了使用同一个寡聚体进行多种基因的诱变，使用者只需量化特定于其诱变实验的寡聚体部分，并调整寡聚体的体积和有效浓度，以用于缺口突变。
[背景] 蛋白质功能序列相关性的评价对于蛋白质科学的应用和基础研究具有重要意义。近年来，深度突变扫描（DMS）已经上升到基于蛋白质的研究的前沿（Fowler and ...
|
|
|