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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 and with a high concentration of polyethylene glycol (PEG). The 3’ capture step enables precise determination of the 3’ ends of diverse RNA molecules. Additionally, a random hexamer in the ligated ...
[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., 2013c; Gianella et al., 2014). This protocol was designed to collect and process male genital secretion (GS), and to isolate and further quantify HIV RNA and DNA of seven HHV from seminal ...
[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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