Author:
Date:
2018-02-20
[Abstract] Next generation high-throughput sequencing has enabled sensitive and unambiguous analysis of RNA populations in cells. Here, we describe a method for isolation and strand-specific sequencing of small RNA pools from bacteria that can be multiplexed to accommodate multiple biological samples in a single experiment. Small RNAs are isolated by polyacrylamide gel electrophoresis and treated with T4 polynucleotide kinase. This allows for 3’ adapter ligation to CRISPR RNAs, which don’t have pre-existing 3’-OH ends. Pre-adenylated adapters are then ligated using T4 RNA ligase 1 in the absence of ATP ...
[摘要] 新一代高通量测序技术能够对细胞中的RNA群体进行敏感和明确的分析。在这里,我们描述了一种从细菌中分离和链特异性测序小RNA池的方法,所述细菌可以在单个实验中多路复用以容纳多个生物样品。小RNA通过聚丙烯酰胺凝胶电泳分离并用T4多核苷酸激酶处理。这允许3'衔接头连接至CRISPR RNA,其不具有预先存在的3'-OH末端。然后使用T4 RNA连接酶1在不存在ATP和高浓度聚乙二醇(PEG)的情况下将前腺苷酸化的衔接子连接。 3'捕获步骤能够精确测定不同RNA分子的3'末端。此外,连接适配器中的随机六聚体有助于控制潜在的下游扩增偏差。逆转录后,将cDNA产物环化并通过PCR制备文库。我们显示扩增的文库不需要通过凝胶电泳可见,以期望产物的有效测序。使用这种方法,我们通常从少量纯化的小RNA制备RNA测序文库。该协议适合于通过对成熟的CRISPR RNA进行测序来测定细菌中的CRISPR RNA生物合成,但可以用于测序不同类型的小RNA。我们还提供了一个完整的数据处理管道示例,并提供了运行所提供脚本的说明。
【背景】与聚集的经常散布的短回文重复序列(CRISPR)相关的遗传模块赋予不同的原核宿主适应性免疫(Barrangou et ...
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Author:
Date:
2015-05-05
[Abstract] Multiple viruses can co-infect the genital tract, modifying the immunologic and virologic milieu and possibly playing a role in viral transmission and pathogenesis. The aim of our studies has been to understand the complex relationships between HIV-1 RNA, and multiple human herpesviruses known to frequently replicate in the genital tract of HIV-infected men (i.e. cytomegalovirus [CMV], Epstein Bar virus [EBV], herpes simplex virus [HSV] types 1 and 2, and human herpesviruses [HHV] 6, 7 and 8) (Gianella et al., 2013a; Gianella et al., 2013b; Gianella et al., ...
[摘要] 多种病毒可以共感染生殖道,修改免疫学和病毒学环境,并可能在病毒传播和发病中起作用。 我们的研究的目的是了解HIV-1 RNA和已知在HIV感染的男性生殖道中经常复制的多种人疱疹病毒之间的复杂关系(即巨细胞病毒[CMV],Epstein 条形病毒[EBV],单纯疱疹病毒[HSV] 1型和2型,以及人疱疹病毒[HHV] 6,7和8)(Gianella等人,2013a; Gianella等人 。,2013b; Gianella 等人,2013c; Gianella ,2014)。 该方案设计用于收集和处理男性生殖器分泌(GS),并使用定量实时PCR技术从精浆中分离并进一步定量7个HHV的HIV RNA和DNA。
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