{{'Search' | translate}}
 

RNaseOUTTM Recombinant Ribonuclease Inhibitor

Company: Invitrogen
Catalog#: 10777019
Bio-protocol()
Company-protocol()
Other protocol()

Primer ID Next-Generation Sequencing for the Analysis of a Broad Spectrum Antiviral Induced Transition Mutations and Errors Rates in a Coronavirus Genome
Author:
Date:
2021-03-05
[Abstract]  

Next generations sequencing (NGS) has become an important tool in biomedical research. The Primer ID approach combined with the MiSeq platform overcomes the limitation of PCR errors and reveals the true sampling depth of population sequencing, making it an ideal tool to study mutagenic effects of potential broad-spectrum antivirals on RNA viruses. In this report we describe a protocol using Primer ID sequencing to study the mutations induced by antivirals in a coronavirus genome from an in vitro cell culture model and an in vivo mouse model. Viral RNA or total lung tissue RNA is tagged with Primer ID-containing cDNA primers during the initial reverse transcription step, followed by two rounds of PCR to amplify viral sequences and incorporate sequencing adaptors. Purified and pooled

...
[摘要]  [摘要]下一代测序(NGS)已成为生物医学研究的重要工具。结合MiSeq平台的Primer ID方法克服了PCR错误的局限性,并揭示了群体测序的真实采样深度,使其成为研究潜在的广谱抗病毒剂对RNA病毒的诱变作用的理想工具。在本报告中,我们描述了一种使用引物ID测序的方案,用于研究体外细胞培养模型和体内小鼠模型中冠状病毒基因组中抗病毒药诱导的突变。在最初的反转录步骤中,病毒RNA或总肺组织RNA用含Primer ID的cDNA引物标记,然后进行两轮PCR扩增病毒序列并整合测序适配器。使用MiSeq平台对纯化和合并的文库进行测序。测序数据使用模板共有序列(TCS)网络应用处理。引物ID方法提供了一种精确的测序方案,可以测量病毒RNA基因组和宿主mRNA中的突变错误率。测序结果表明,β-D-N4-羟基胞嘧啶核苷(NHC)大大提高了病毒RNA基因组中的过渡取代率,但并未显着提高颠覆取代率,并且发现胞嘧啶(C)至尿苷(U)是最常见的突变。


[背景]下一代测序(NGS)已被广泛应用在生物医学研究中使用在过去十年。当应用NGS研究宿主内病毒种群的RNA病毒时,需要考虑对文库制备和测序方案的修改。样本之间的病毒滴度(或病毒载量)差异很大。传统的NGS平台在测序运行中需要1-500 ng的DNA(或RNA),但在大多数情况下,临床样品中的病毒RNA少于100 ...

Detecting Spaciotemporal Transcript Accumulation in Maize by RNA In Situ Hybridization
Author:
Date:
2021-02-20
[Abstract]  RNA in situ hybridization is a method for visualizing spatiotemporal transcript accumulation in cells and tissues. The method provides clear resolution, is highly sensitive and specific, and can uncover gradients of transcript accumulation within a histologically-intact tissue, which is not possible currently with other methods for transcript detection. RNA in situ hybridization, however, is not a quantitative approach for gene expression. Protocols for RNA in situ hybridization have numerous steps that can span several days of work, complicating troubleshooting procedures. Here, we build on previously published RNA in situ hybridization protocols optimized for paraffin-embedded and sectioned maize tissue (Jackson, 1991; Long et al., 1996 ; ... [摘要]  [摘要] RNA原位杂交是一种可视化时空转录本在细胞和组织中积累的方法。该方法提供清晰的分辨率,高度灵敏和特异,并且可以揭示组织完整的组织内转录物积累的梯度,这是当前其他方法无法检测到转录物的方法。然而,RNA原位杂交不是用于基因表达的定量方法。RNA原位杂交的协议有许多步骤,可能需要花费数天的时间,从而使故障排除过程变得更加复杂。在这里,我们基于先前发布的RNA原位构建 杂交方案针对石蜡包埋和切片的玉米组织进行了优化(Jackson,1991; Long等,1996;Javelle等,2011),它提供了优化转录本检测的其他措施。


图形摘要:


RNA原位杂交的工作流程



[背景技术]在时间和空间差异基因表达允许具有相同的遗传物质的细胞呈现不同的身份。确实,基因表达的这种变化通常负责驱动整个生物体中模式或形态的进化变化(Carroll,2005)。因此,通过观察组织内天然背景下的基因表达,可以增强对发育过程的见识。诸如RT-qPCR和RNA- ...

DigiTAG–a RNA Sequencing Approach to Analyze Transcriptomes of Rare Cell Populations in Drosophila melanogaster
Author:
Date:
2020-11-05
[Abstract]  

Cell-type specific transcriptional programs underlie the development and maintenance of organs. Not only distinct cell types within a tissue, even cells with supposedly identical cell fates show a high degree of transcriptional heterogeneity. Inevitable, low cell numbers are a major hurdle to study transcriptomes of pure cell populations. Here we describe DigiTAG, a high-throughput method that combines transposase fragmentation and molecular barcoding to retrieve high quality transcriptome data of rare cell types in Drosophila melanogaster. The protocol showcases how DigiTAG can be used to analyse the transcriptome of rare neural stem cells (type II neuroblasts) of Drosophila larval brains, but can also be utilized for other cell types or model systems.

[摘要]  [摘要]细胞类型的特定转录程序是器官的发育和维持的基础。不仅组织内不同的细胞类型,甚至具有相同细胞命运的细胞也显示出高度的转录异质性。不可避免的是,低细胞数量是研究纯细胞群体转录组的主要障碍。在这里,我们介绍DigiTAG ,这是一种高通量方法,将转座酶片段化和分子条形码相结合,以检索果蝇中稀有细胞类型的高质量转录组数据。该协议展示了DigiTAG如何可用于分析果蝇幼虫的罕见神经干细胞(II型成神经细胞)的转录组 大脑,但也可以用于其他细胞类型或模型系统。

[背景]在发育过程中,不同细胞类型之间的过渡与组织稳态之间的关系是由大量转录因子及其诱导的转录变化所精心安排的。在过去的十年中,RNA测序(RNA- seq )已成为测量整个基因组转录动力学的经典方法(Stark等,2019)。组织上的大量RNA序列不允许研究不同细胞群体的转录网络,特别是稀有细胞类型的转录网络。因此,需要提供低输入样品高质量转录组的RNA- seq方案。

在果蝇中,有限的材料通常构成分析特定组织或细胞类型的障碍。果蝇神经干细胞(称为神经母细胞)很好地说明了这一点(Homem和Knoblich ,2012)。存在成神经细胞的几个不同的亚群。例如,在果蝇的幼虫大脑中,只有16种II型成神经细胞产生神经元,神经元支配了运动和感觉处理所需的大脑区域(Walsh and ...

Comments