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DynaMag-2 Magnet

DynaMag TM -2 Magnet

Company: Thermo Fisher Scientific
Catalog#: 12321D
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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

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[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 ...

Native Co-immunoprecipitation Assay to Identify Interacting Partners of Chromatin-associated Proteins in Mammalian Cells
Author:
Date:
2020-12-05
[Abstract]  

Protein-protein interactions play key roles in nuclear processes including transcription, replication, DNA damage repair, and recombination. Co-immunoprecipitation (Co-IP) followed by western blot or mass spectrometry is an invaluable approach to identify protein-protein interactions. One of the challenges in the Co-IP of a protein localized to nucleus is the extraction of nuclear proteins from sub-nuclear fractions without losing physiologically relevant protein interactions. Here we describe a protocol for native Co-IP, which was originally used to successfully identify previously known as well novel topoisomerase 1 (TOP1) interacting proteins. In this protocol, we first extracted nuclear proteins by sequentially increasing detergent and salt concentrations, the extracted fractions were

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[摘要]  [摘要]蛋白质间相互作用 在核过程中起关键作用,包括转录,复制,DNA损伤修复和重组。免疫共沉淀(Co-IP),然后进行蛋白质印迹或质谱分析是鉴定蛋白质-蛋白质相互作用的宝贵方法。在Co-IP中定位于细胞核的蛋白质中的挑战之一是从亚核级分中提取核蛋白质,而又不会失去生理上相关的蛋白质相互作用。在这里,我们描述了一种用于天然Co-IP的协议,该协议最初用于成功地识别以前称为新拓扑拓扑异构酶1(TOP1)相互作用的蛋白质。在此协议中,我们首先通过依次增加去污剂和盐浓度来提取核蛋白,然后将提取的级分稀释,合并并用于Co-IP。该协议可用于鉴定多种哺乳动物细胞中其他染色质相关蛋白的蛋白相互作用组。


背景]钴- IP被广泛地被使用,以解开的错综复杂的关系之间的蛋白复合物和各种染色质交易期间的复制,转录,和基因组的维护。但是,它是具有挑战性的,以保持不稳定的蛋白质-蛋白质相互作用的完整过程中提取,免疫沉淀和一个共同的IP实验的洗涤步骤。稳定不稳定蛋白质相互作用的一种方法是在细胞裂解之前用细胞可渗透的可逆化学交联剂(例如丙酸二硫代双琥珀酰亚胺酯)处理细胞(Smith等人,2011)。由于该方法伴随着诸如提取效率低和非特异性蛋白质捕获之类的缺点,因此优选不交联的Co-IP(天然IP)。

一核蛋白质可以被分配到不同的子-核舱或染色质区域是需要不同程度的严格性为它的提取和溶解。对于例如,TOP ...

Whole-genome Identification of Transcriptional Start Sites by Differential RNA-seq in Bacteria
Author:
Date:
2020-09-20
[Abstract]  Gene transcription in bacteria often starts some nucleotides upstream of the start codon. Identifying the specific Transcriptional Start Site (TSS) is essential for genetic manipulation, as in many cases upstream of the start codon there are sequence elements that are involved in gene expression regulation. Taken into account the classical gene structure, we are able to identify two kinds of transcriptional start site: primary and secondary. A primary transcriptional start site is located some nucleotides upstream of the translational start site, while a secondary transcriptional start site is located within the gene encoding sequence.

Here, we present a step by step protocol for genome-wide transcriptional start sites determination by differential RNA-sequencing (dRNA-seq) ...
[摘要]  [摘要] 细菌中的基因转录通常起始于起始密码子上游的一些核苷酸。识别SPE cific Ť ranscriptional 小号挞小号ITE (TSS)为遗传操作必需的,因为在许多情况下,起始密码子上游有中涉及的基因表达调控序列元件。考虑到经典的基因结构,我们能够鉴定出两种转录起始位点:一级和二级。主要转录起始位点位于翻译起始位点上游的一些核苷酸上,而次要转录起始位点位于基因编码序列内。

这里,我们提出一步步协议全基因组吨ranscriptional 小号馅饼小号ITES d etermination通过差RNA测序(DRNA 使用肠道病原体-SEQ)福氏痢疾杆菌血清型菌株5A作为M90T模型。但是,该方法可以用于选择的任何其他细菌物种。第一步,使用热酚法从细菌培养物中纯化总RNA。核糖体RNA(rRNA)是使用商业试剂盒通过杂交探针特异性去除的。然后准备一个富含5'- 一磷酸依赖性核酸外切酶(TEX)处理的,富含初级转录本的RNA文库,用于与未进行TEX处理的文库进行比较,然后连接已知序列的RNA接头衔接子,从而确定具有单核苷酸精度的TSS。最后,对RNA进行处理以制备Illumina测序文库,并按购买的服务进行测序。通过内部生物信息学分析鉴定TSS。

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