{{'Search' | translate}}
 

PCR Thermal Cycler

Company: Thermo Fisher Scientific
Catalog#: 4484075
Bio-protocol()
Company-protocol()
Other protocol()

EmPC-seq: Accurate RNA-sequencing and Bioinformatics Platform to Map RNA Polymerases and Remove Background Error
Author:
Date:
2021-02-20
[Abstract]  

Transcription errors can substantially affect metabolic processes in organisms by altering the epigenome and causing misincorporations in mRNA, which is translated into aberrant mutant proteins. Moreover, within eukaryotic genomes there are specific Transcription Error-Enriched genomic Loci (TEELs) which are transcribed by RNA polymerases with significantly higher error rates and hypothesized to have implications in cancer, aging, and diseases such as Down syndrome and Alzheimer’s. Therefore, research into transcription errors is of growing importance within the field of genetics. Nevertheless, methodological barriers limit the progress in accurately identifying transcription errors. Pro-Seq and NET-Seq can purify nascent RNA and map RNA polymerases along the genome but cannot be

...
[摘要]  [摘要]转录错误可通过改变表观基因组并引起mRNA的错误整合而严重影响生物体内的代谢过程,从而将其翻译为异常的突变蛋白。此外,真核基因组内有特定转录错误富集的基因组基因座(TEELs),它们由RNA聚合酶与显著更高的错误率转录并推测为具有影响在癌症,老化和疾病例如唐氏综合征和阿尔茨海默'秒。因此,在遗传学领域对转录错误的研究越来越重要。尽管如此,方法上的障碍限制了准确识别转录错误的进展。Pro-Seq和NET-Seq可以沿基因组纯化新生RNA并绘制RNA聚合酶,但不能用于鉴定转录突变。在这里,我们本背景误差模型耦合的精密核圆形测序上运行(EMPC -SEQ),一种方法COMBIN荷兰国际集团测定和圆形测序核上运行与背景误差模型精确地检测新生转录错误和有效地辨别TEELs基因组中。

[背景]核糖核苷酸错掺导致的转录错误在所有活生物体中无处不在(Carey,2015)。假设每个信使RNA(mRNA)可以翻译2-4千次(Schwanhausser et al。,2011),并且许多特殊RNA在给定时间每个细胞仅表达一次(Islam et al。,2011; Pelechano et al。,2011)。,2010),即使是关键残基的单个转录错误也会使特定蛋白质的表达产生很大差异。另外,转录错误可加速蛋白质聚集,导致人类中与年龄有关的疾病(van ...

Multiplex T-cell Stimulation Assay Utilizing a T-cell Activation Reporter-based Detection System
Author:
Date:
2021-01-20
[Abstract]  

Immune tolerance and response are both largely driven by the interactions between the major histocompatibility complex (MHC) expressed by antigen presenting cells (APCs), T-cell receptors (TCRs) on T-cells, and their cognate antigens. Disordered interactions cause the pathogenesis of autoimmune diseases such as type 1 diabetes. Therefore, the identification of antigenic epitopes of autoreactive T-cells leads to important advances in therapeutics and biomarkers. Next-generation sequencing methods allow for the rapid identification of thousands of TCR clonotypes from single T-cells, and thus there is a need to determine cognate antigens for identified TCRs. This protocol describes a reporter system of T-cell activation where the fluorescent reporter protein ZsGreen-1 is driven by nuclear

...
[摘要]  [摘要] 免疫耐受和应答都很大程度上由抗原呈递细胞(APC)表达的主要组织相容性复合物(MHC),T细胞上的T细胞受体(TCR)及其同源抗原之间的相互作用驱动。相互作用障碍导致自身免疫性疾病(例如1型糖尿病)的发病机理。因此,鉴定自身反应性T细胞的抗原表位导致治疗和生物标志物的重要进展。下一代测序方法可从单个T细胞快速鉴定数千种TCR克隆型,因此需要确定已鉴定TCR的同源抗原。该协议描述了T细胞活化的报告系统,其中荧光报告蛋白ZsGreen-1由活化T细胞的核因子(NFAT)信号驱动并通过流式细胞仪读取。记者T细胞也组成性表达额外的一对荧光素tein作为识别物,允许同时多路复用多达8种不同的报告T细胞系,每种表达不同的目标TCR,可通过流式细胞仪区分。一旦制成TCR表达细胞系,仅需一个转导步骤即可将其无限期用于制备新的T细胞系。这种多路复用系统允许筛选TCR-抗原相互作用的数量,否则这些相互作用将是不切实际的,可在多种情况下使用(即,筛选单个抗原或抗原库),并可用于研究任何T细胞-MHC-抗原三分子相互作用。

[背景] T细胞,抗原呈递细胞(APC)及其同源抗原之间的相互作用是自身免疫性疾病(例如1型糖尿病)的主要事件(Michels等,2017; ...

Comments