Tethered Chromosome Conformation Capture Sequencing in Triticeae: A Valuable Tool for Genome Assembly
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Author:
Date:
2018-08-05
[Abstract] Chromosome conformation capture sequencing (Hi-C) is a powerful method to comprehensively interrogate the three-dimensional positioning of chromatin in the nucleus. The development of Hi-C can be traced back to successive increases in the resolution and throughput of chromosome conformation capture (3C) (Dekker et al., 2002). The basic workflow of 3C consists of (i) fixation of intact chromatin, usually by formaldehyde, (ii) cutting the fixed chromatin with a restriction enzyme, (iii) religation of sticky ends under diluted conditions to favor ligations between cross-linked fragments or those between random fragments and (iv) quantifying the number of ligations events between pairs of genomic loci (de Wit and de Laat, 2012). In the original 3C protocol, ligation frequency was ...
[摘要] 染色体构象捕获测序(Hi-C)是一种全面询问细胞核中染色质三维定位的有效方法。 Hi-C的发展可以追溯到染色体构象捕获的分辨率和通量的连续增加(3C)(Dekker et al。,2002)。 3C的基本工作流程包括(i)通常用甲醛固定完整的染色质,(ii)用限制酶切割固定的染色质,(iii)在稀释条件下重新连接粘性末端,以促进交联片段之间的连接或随机片段之间的那些和(iv)量化基因组基因座对之间的连接事件的数量(de Wit和de Laat,2012)。在最初的3C方案中,通过半定量PCR扩增对应于少量基因组位点(“一对一”)的选定连接接头来测量连接频率(Dekker et al。,2002 )。然后,染色体构象捕获芯片(4C)和染色体构象捕获碳复制(5C)技术扩展3C以分别以“一对多”或“多对多”方式计算结扎事件。 Hi-C(Lieberman-Aiden et al。,2009)最终将3C与下一代测序相结合(Metzker,2010)。此处,在再连接之前,用生物素标记的核苷酸类似物填充粘性末端以在后续步骤中富集具有连接连接的片段。然后对Hi-C文库进行高通量测序,并将得到的读数映射到参考基因组,允许以“多对多”方式确定接触概率,其分辨率仅受限制性位点的分布限制和阅读深度。 Hi-C的首次应用是阐明人类基因组中的全球染色质折叠原理(Lieberman-Aiden et ...
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In vitro NLK Kinase Assay
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Author:
Date:
2017-11-05
[Abstract] This protocol provides step by step instructions to perform an in vitro kinase assay for nemo-like kinase. In addition, this protocol also describes an efficient method using mild lysis buffer for expression and purification of Glutathione S-transferase (GST) fusion proteins.
[摘要] 该协议提供了一步一步的指导,以进行nemo样激酶的体外激酶测定。 此外,该方案还描述了使用温和裂解缓冲液来表达和纯化谷胱甘肽S-转移酶(GST)融合蛋白的有效方法。
【背景】果蝇nemo已经被确定为在发育中的果蝇眼中光感受器簇的关键调节剂(Choi和Benzer,1994)。 据报道,nemo是不同物种间保守的。 该哺乳动物同系物是类似于Nemo的激酶(NLK)(Brott等人,1998; Rocheleau等人,1999)。 NLK是CMGC集团(CDK,MAPK,GSK3和CLK)的成员,也属于非典型的MAPK家族。 NLK通过转录因子调节多种细胞机制,所述转录因子包括在不同信号传导途径中的关键参与者TCF / LEF1,NICD和FOXO(Ishitani等人,1999; Ishitani等人, ,2010; Kim等人,2010)。 此外,最近我们已经将NLK鉴定为Yes相关蛋白(YAP)的新型激酶,YAP是Hippo途径中的共转录激活剂(Moon等人,2017)。 该协议描述了用于测量类似尼莫地来激酶活性的体外方法。
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Expression and Purification of a Mammalian P2X7 Receptor from Sf9 Insect Cells
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Author:
Date:
2017-09-05
[Abstract] The P2X7 receptor is an extracellular ATP-gated ion channel found only in eukaryotes (Bartlett et al., 2014). Due to its unique properties among P2X receptors, such as formation of a large conductance pore, the P2X7 receptor has been implicated in devastating diseases like chronic pain (North and Jarvis, 2013). However, mechanisms underlying the P2X7 specific properties remain poorly understood, partly because purification of this eukaryotic membrane protein has been challenging. Here we describe a detailed protocol for expressing and purifying a mammalian P2X7 receptor using an insect cell-baculovirus system. The P2X7 receptor is expressed in Sf9 insect cells as a GFP fusion protein and solubilized with a buffer containing Triton X-100 detergent. The P2X7-GFP fusion protein is ...
[摘要] P2X7受体是仅在真核生物中发现的胞外ATP门控离子通道(Bartlett等,2014)。由于其P2X受体之间的独特性质,例如大电导孔的形成,P2X7受体已经涉及破坏性疾病如慢性疼痛(North和Jarvis,2013)。然而,P2X7特异性属性的机制仍然知之甚少,部分原因是纯化这种真核膜蛋白是一个挑战。在这里,我们描述了使用昆虫细胞 - 杆状病毒系统表达和纯化哺乳动物P2X7受体的详细方案。 P2X7受体在作为GFP融合蛋白的Sf9昆虫细胞中表达,并用含有Triton X-100洗涤剂的缓冲液溶解。然后使用Strep-Tactin亲和层析在含有十二烷基麦芽糖苷的缓冲液中纯化P2X7-GFP融合蛋白。在通过凝血酶酶切割连接的GFP和Strep-标签后,使用大小排阻色谱分离P2X7受体。该方法通常从6L的Sf9培养物产生约2mg的纯化蛋白质。纯化的蛋白质可以用含有15%甘油的缓冲液在4℃下储存至少2个月,并用于各种功能和结构研究(Karasawa和Kawate,2016)。 【背景】P2X7受体是嘌呤能P2X受体家族的七种亚型之一,并且是广泛疾病如神经退行性疾病,癫痫和神经性疼痛的有希望的新型药物靶点(North和Jarvis,2013; Bhattacharya和Biber, ...
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