| EmPC-seq: Accurate RNA-sequencing and Bioinformatics Platform to Map RNA Polymerases and Remove Background Error
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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 ...
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| Visualization of RNA 3’ ends in Escherichia coli Using 3’ RACE Combined with Primer Extension
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Author:
Date:
2018-03-05
[Abstract] In this assay, 3’ RACE (Rapid Amplification of cDNA 3’ Ends) followed by PE (primer extension), abbreviated as 3’ RACE-PE is used to identify the mRNA 3’ ends. The following protocol describes the amplification of the mRNA 3’ ends at the galactose operon in E. coli and the corresponding visualization of the PCR products through PE. In PE, the definite primer is 5’ end-labeled using [γ-(32) P] ATP and T4 polynucleotide kinase, which anneals to the specific DNA molecules within the PCR product of the 3’ RACE. The conventional PE can only be used to locate the 5’ end of an mRNA transcript since reverse transcriptase (RTase) polymerizes only in the 5’ → 3’ direction. Thus, Taq polymerase is used instead of RTase, PCR is performed. Therefore, we are able to locate the 3’ end of the ...
[摘要] 在该测定中，使用缩写为3'RACE-PE的3'RACE（cDNA3'末端快速扩增），随后是PE（引物延伸），以鉴定mRNA3'末端。以下方案描述E中半乳糖操纵子的mRNA 3'末端的扩增。并通过PE对相应的PCR产物进行可视化。在PE中，使用[γ-（32）P] ATP和T4多核苷酸激酶对确定的引物进行5'末端标记，其退火至3'RACE的PCR产物内的特定DNA分子。由于逆转录酶（RTase）仅在5'→3'方向聚合，常规PE只能用于定位mRNA转录物的5'末端。因此，使用Taq聚合酶代替RTase，进行PCR。因此，我们能够使用此测定法定位mRNA的3'末端。通过在变性8％尿素-PAGE（聚丙烯酰胺凝胶电泳）凝胶中分离DNA产物，可以直接显示和定量3'末端的相对量。 3'末端的确切位置可以通过比较这些最终的DNA产物与相应的DNA测序阶梯进行测序。
【背景】mRNA 3'末端的合成是E中的重要步骤。产生稳定的信使RNA（mRNA）的大肠杆菌。在真核细胞中，mRNA 3'末端形成是通过从内部磷酸二酯键切割，然后加入聚（A）尾;而在原核细胞中，通过终止转录或通过加工初级转录产生mRNA的3'末端（Altman和Robertson，1973; Nudler和Gottesman，2002; Zhao等人，1999年）。因此，分析mRNA ...
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| Preparation of Multiplexed Small RNA Libraries from Plants
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Author:
Date:
2014-11-05
[Abstract] High-throughput sequencing is a powerful tool for exploring small RNA populations in plants. The ever-increasing output from an Illumina Sequencing System allows for multiplexing multiple samples while still obtaining sufficient data for small RNA discovery and characterization. Here we describe a protocol for generating multiplexed small RNA libraries for sequencing up to 12 samples in one lane of an Illumina HiSeq System single-end, 50 base pair run. RNA ligases are used to add the 3’ and 5’ adaptors to purified small RNAs; ligation products that lack a small RNA molecule (adaptor-adaptor products) are intentionally depleted. After cDNA synthesis, a linear PCR step amplifies the DNA fragments. The 3’ PCR primers used here include unique 6-nucleotide sequences to allow for multiplexing ...
[摘要] 高通量测序是探索植物中小RNA群体的强大工具。 来自Illumina测序系统的不断增加的输出允许多重多个样品，同时仍然获得足够的数据用于小RNA发现和表征。 在这里我们描述了一个协议，用于生成多重小RNA文库，用于在Illumina HiSeq系统单端，50碱基对运行的一个泳道中测序多达12个样品。 RNA连接酶用于将3'和5'接头添加至纯化的小RNA; 缺少小RNA分子（衔接子 - 衔接子产物）的连接产物被故意耗尽。 cDNA合成后，线性PCR步骤扩增DNA片段。 本文使用的3'PCR引物包括独特的6-核苷酸序列，以允许多达多达12个样品。
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