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Diethyl pyrocarbonate (DEPC)

Company: Sigma
Catalog#: 40718
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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

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[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 ...

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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