| 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 ...
|
|
|
| Generation of Gene Knockout and Gene Replacement with Complete Removal of Full-length Endogenous Transcript Using CRISPR-Trap
|
|
Author:
Date:
2018-10-20
[Abstract] This protocol describes the application of the CRISPR-Trap from designing of the gene targeting strategy to validation of successfully edited clones that was validated on various human cell lines, among them human induced pluripotent stem cells (hiPSCs). The advantage of CRISPR-Trap over conventional approaches is the complete removal of any endogenous full-length transcript from the target gene. CRISPR-Trap is applicable for any target gene with no or little coding sequence in its first exon. Several human cell lines and different genes have so far been edited successfully with CRISPR-Trap.
[摘要] 该协议描述了CRISPR-Trap从设计基因靶向策略到验证成功编辑的克隆的应用，所述克隆在各种人细胞系上得到验证，其中人类诱导的多能干细胞（hiPSC）。 CRISPR-Trap优于常规方法的优点是从靶基因完全去除任何内源全长转录物。 CRISPR-Trap适用于在其第一个外显子中没有编码序列或编码序列很少的任何靶基因。 到目前为止，已经使用CRISPR-Trap成功编辑了几种人细胞系和不同基因。
【背景】CRISPR / Cas9技术的出现促进了基因敲除和基因编辑的基因组靶向。执行敲除的常规方法依赖于引入移码导致过早终止密码子（PTC），截短开放阅读框（ORF）以及随后通过无义介导的mRNA衰变（NMD）降解靶基因的转录物。 。这种方法的一个可能的缺陷是全长转录物，其可以逃避NMD并产生具有残余或甚至显性负功能的C末端截短蛋白。该协议提出了CRISPR-Trap，这是我们最近建立的一种方法（Reber et al。>，2018），成功编辑后将阻止从靶基因位点表达任何全长转录本（图1）。简而言之，这种方法针对CRISPR / ...
|
|
|