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Current Protocols in Molecular Biology最新文献

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RibORF: Identifying Genome-Wide Translated Open Reading Frames Using Ribosome Profiling RibORF:利用核糖体分析鉴定全基因组翻译开放阅读框
Current Protocols in Molecular Biology Pub Date : 2018-09-04 DOI: 10.1002/cpmb.67
Zhe Ji
{"title":"RibORF: Identifying Genome-Wide Translated Open Reading Frames Using Ribosome Profiling","authors":"Zhe Ji","doi":"10.1002/cpmb.67","DOIUrl":"10.1002/cpmb.67","url":null,"abstract":"<p>Ribosome profiling identifies RNA fragments associated with translating ribosomes. The technology provides an opportunity to examine genome-wide translation events at single-nucleotide resolution and in an unbiased manner. Here I present a computational pipeline named RibORF to systematically identify translated open reading frames (ORFs), based on read distribution features representing active translation, including 3-nt periodicity and uniformness across codons. Analyses using the computational tool revealed pervasive translation in putative ‘noncoding’ regions, such as lncRNAs, pseudogenes, and 5′UTRs. The computational tool is useful for studying functional roles of non-canonical translation events in various biological processes. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10734,"journal":{"name":"Current Protocols in Molecular Biology","volume":"124 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmb.67","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36455316","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 24
Rfoot: Transcriptome-Scale Identification of RNA-Protein Complexes from Ribosome Profiling Data 从核糖体分析数据中鉴定rna -蛋白复合物的转录组尺度
Current Protocols in Molecular Biology Pub Date : 2018-08-31 DOI: 10.1002/cpmb.66
Zhe Ji
{"title":"Rfoot: Transcriptome-Scale Identification of RNA-Protein Complexes from Ribosome Profiling Data","authors":"Zhe Ji","doi":"10.1002/cpmb.66","DOIUrl":"10.1002/cpmb.66","url":null,"abstract":"<p>Ribosome profiling was developed to identify genome-wide RNA fragments associated with translating ribosomes. However, no experimental procedure was developed to effectively purify ribosome-RNA complexes. Actually, ribosome profiling is a transcriptomic RNase footprinting assay, which can identify both ribosomal and non-ribosomal protein-RNA complexes. Many sequencing reads represent functional RNA footprints associated with non-ribosomal proteins. Here I present a computational pipeline named Rfoot to systematically identify genome-wide non-ribosomal RNA footprints, based on the highly localized read distribution feature. Analyses have revealed native functional protein-RNA complexes in lncRNAs, 3′UTRs of mRNAs, and all types of small noncoding RNAs. This computational tool is useful for discovering novel noncoding functions of RNAs. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10734,"journal":{"name":"Current Protocols in Molecular Biology","volume":"124 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-08-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmb.66","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36450620","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 8
T7 Expression Systems for Inducible Production of Proteins from Cloned Genes in E. coli 克隆基因在大肠杆菌中诱导产蛋白的T7表达系统
Current Protocols in Molecular Biology Pub Date : 2018-07-17 DOI: 10.1002/cpmb.63
F. William Studier
{"title":"T7 Expression Systems for Inducible Production of Proteins from Cloned Genes in E. coli","authors":"F. William Studier","doi":"10.1002/cpmb.63","DOIUrl":"10.1002/cpmb.63","url":null,"abstract":"<p>Inducible T7 expression systems are capable of producing a wide range of proteins in <i>E. coli</i>. Improvements over common practice include: (1) preventing unintended induction by establishing and maintaining expression strains in non-inducing growth media composed entirely of purified components instead of complex growth media that may variably induce target proteins on approach to saturation; and (2) expressing many target proteins in parallel by convenient and productive auto-induction in BL21(DE3) and other suitable hosts, instead of IPTG induction. From the earliest days, basal expression prevented establishment of inducible strains for producing proteins that are stressful to the host. Newly developed pAL vectors now reduce basal expression to levels where coding sequences for even the most stressful proteins can be maintained and induced. Asymmetric ligation allows simple and efficient cloning of individual coding sequences or simultaneous cloning of two or three coding sequences for co-expression from a single pAL vector. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10734,"journal":{"name":"Current Protocols in Molecular Biology","volume":"124 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-07-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmb.63","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36320917","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 15
Expression and Purification of Recombinant Proteins Using the Baculovirus System 杆状病毒系统中重组蛋白的表达与纯化
Current Protocols in Molecular Biology Pub Date : 2018-06-28 DOI: 10.1002/cpmb.61
Cheryl Isaac Murphy, Helen Piwnica-Worms, Stefan Grünwald, William G. Romanow, Nicole Francis, Hua-Ying Fan, Sharon Marr
{"title":"Expression and Purification of Recombinant Proteins Using the Baculovirus System","authors":"Cheryl Isaac Murphy,&nbsp;Helen Piwnica-Worms,&nbsp;Stefan Grünwald,&nbsp;William G. Romanow,&nbsp;Nicole Francis,&nbsp;Hua-Ying Fan,&nbsp;Sharon Marr","doi":"10.1002/cpmb.61","DOIUrl":"10.1002/cpmb.61","url":null,"abstract":"<p>This article describes how to analyze protein expression in cells infected with recombinant baculovirus on a small scale for optimizing protein production, how to maximize and scale up recombinant protein production, and how to purify recombinant proteins. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10734,"journal":{"name":"Current Protocols in Molecular Biology","volume":"123 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-06-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmb.61","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36266685","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
CAPTURE: In Situ Analysis of Chromatin Composition of Endogenous Genomic Loci by Biotinylated dCas9 捕获:用生物素化dCas9原位分析内源性基因组位点的染色质组成
Current Protocols in Molecular Biology Pub Date : 2018-06-19 DOI: 10.1002/cpmb.64
Xin Liu, Yuannyu Zhang, Yong Chen, Mushan Li, Zhen Shao, Michael Q. Zhang, Jian Xu
{"title":"CAPTURE: In Situ Analysis of Chromatin Composition of Endogenous Genomic Loci by Biotinylated dCas9","authors":"Xin Liu,&nbsp;Yuannyu Zhang,&nbsp;Yong Chen,&nbsp;Mushan Li,&nbsp;Zhen Shao,&nbsp;Michael Q. Zhang,&nbsp;Jian Xu","doi":"10.1002/cpmb.64","DOIUrl":"10.1002/cpmb.64","url":null,"abstract":"<p><i>Cis</i>-regulatory elements (CREs) play a pivotal role in spatiotemporal control of tissue-specific gene expression, yet the molecular composition of the vast majority of CREs in native chromatin remains unknown. In this article, we describe the clustered regularly interspaced short palindromic repeats (CRISPR) affinity purification <i>in situ</i> of regulatory elements (CAPTURE) approach to simultaneously identify locus-specific chromatin-regulating protein complexes and long-range DNA interactions. Using an <i>in vivo</i> biotinylated nuclease-deficient Cas9 (dCas9) protein and programmable single guide RNAs (sgRNAs), this approach allows for high-resolution and locus-specific isolation of protein complexes and long-range chromatin looping associated with single copy CREs in mammalian cells. Unbiased analysis of the compositional structure of developmentally regulated or disease-associated CREs identifies new features of transcriptional regulation. Hence, CAPTURE provides a versatile platform to study genomic locus-regulating chromatin composition in a mammalian genome. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10734,"journal":{"name":"Current Protocols in Molecular Biology","volume":"123 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-06-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmb.64","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36244358","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 11
DMS-seq for In Vivo Genome-Wide Mapping of Protein-DNA Interactions and Nucleosome Centers DMS-seq用于蛋白质- dna相互作用和核小体中心的体内全基因组定位
Current Protocols in Molecular Biology Pub Date : 2018-06-15 DOI: 10.1002/cpmb.60
Taichi Umeyama, Takashi Ito
{"title":"DMS-seq for In Vivo Genome-Wide Mapping of Protein-DNA Interactions and Nucleosome Centers","authors":"Taichi Umeyama,&nbsp;Takashi Ito","doi":"10.1002/cpmb.60","DOIUrl":"10.1002/cpmb.60","url":null,"abstract":"<p>The genome exerts its functions through interactions with proteins. Hence, comprehensive identification of protein-occupied sites by genomic footprinting is critical to an in-depth understanding of genome functions. This unit describes the protocol of dimethyl sulfate-sequencing (DMS-seq). DMS is an alkylating reagent that methylates guanine and adenine in double-stranded DNA. DMS added to the culture medium readily enters the cell and methylates its DNA throughout the genome except for the regions bound by proteins, thereby obviating the need for nuclear isolation in genomic footprinting. Polyamine/AP-endonuclease treatment of DNA isolated from DMS-treated cells induces cleavages at the methylated sites. Deep sequencing of these fragments identifies protein-bound sites as peaks of protected fragments or troughs of cleavage sites. Furthermore, DMS displays an unexpected preference to nucleosome centers, enabling their direct detection without genetic manipulation. Therefore, DMS-seq provides a unique method for non-targeted profiling of in vivo protein-DNA interactions. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10734,"journal":{"name":"Current Protocols in Molecular Biology","volume":"123 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-06-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmb.60","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36243977","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 4
An Effective Recombinant Protein Expression and Purification System in Saccharomyces cerevisiae 一种有效的酿酒酵母重组蛋白表达与纯化体系
Current Protocols in Molecular Biology Pub Date : 2018-06-05 DOI: 10.1002/cpmb.62
Ying Xie, Xiao Han, Yansong Miao
{"title":"An Effective Recombinant Protein Expression and Purification System in Saccharomyces cerevisiae","authors":"Ying Xie,&nbsp;Xiao Han,&nbsp;Yansong Miao","doi":"10.1002/cpmb.62","DOIUrl":"10.1002/cpmb.62","url":null,"abstract":"<p>The expression and purification of recombinant proteins using bacterial vectors is a mature and preferred system to obtain folded and stable proteins. However, functional post-translational protein modifications, such as glycosylation or phosphorylation, can only be achieved using eukaryotic expression systems. In addition, insolubility is another challenge when using proteins expressed in <i>Escherichia coli</i>, such as certain intrinsically disordered proteins, which are more prone to aggregation than folded proteins. Eukaryotic protein expression systems, including human cells, baculovirus/insect cells, and yeast, have become indispensable for the production of functional eukaryotic proteins. This article describes a detailed protocol for performing cytosolic protein expression, protein purification, and protein characterization using the budding yeast <i>Saccharomyces cerevisiae</i>. The introduced protein expression and purification system in yeast are advantageous due to the low cost, high yield, high protein solubility, and minimal expertise required. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10734,"journal":{"name":"Current Protocols in Molecular Biology","volume":"123 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-06-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmb.62","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36243498","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 14
Issue Information TOC 发布信息TOC
Current Protocols in Molecular Biology Pub Date : 2018-06-01 DOI: 10.1111/jssr.12355
{"title":"Issue Information TOC","authors":"","doi":"10.1111/jssr.12355","DOIUrl":"https://doi.org/10.1111/jssr.12355","url":null,"abstract":"","PeriodicalId":10734,"journal":{"name":"Current Protocols in Molecular Biology","volume":"31 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"73621988","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Introduction to Single-Cell RNA Sequencing 单细胞RNA测序简介
Current Protocols in Molecular Biology Pub Date : 2018-04-16 DOI: 10.1002/cpmb.57
Thale Kristin Olsen, Ninib Baryawno
{"title":"Introduction to Single-Cell RNA Sequencing","authors":"Thale Kristin Olsen,&nbsp;Ninib Baryawno","doi":"10.1002/cpmb.57","DOIUrl":"10.1002/cpmb.57","url":null,"abstract":"<p>During the last decade, high-throughput sequencing methods have revolutionized the entire field of biology. The opportunity to study entire transcriptomes in great detail using RNA sequencing (RNA-seq) has fueled many important discoveries and is now a routine method in biomedical research. However, RNA-seq is typically performed in “bulk,” and the data represent an average of gene expression patterns across thousands to millions of cells; this might obscure biologically relevant differences between cells. Single-cell RNA-seq (scRNA-seq) represents an approach to overcome this problem. By isolating single cells, capturing their transcripts, and generating sequencing libraries in which the transcripts are mapped to individual cells, scRNA-seq allows assessment of fundamental biological properties of cell populations and biological systems at unprecedented resolution. Here, we present the most common scRNA-seq protocols in use today and the basics of data analysis and discuss factors that are important to consider before planning and designing an scRNA-seq project. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10734,"journal":{"name":"Current Protocols in Molecular Biology","volume":"122 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-04-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmb.57","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36179399","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 95
Using Barcoded HIV Ensembles (B-HIVE) for Single Provirus Transcriptomics 使用HIV条形码集合(B-HIVE)进行单个原病毒转录组学
Current Protocols in Molecular Biology Pub Date : 2018-04-16 DOI: 10.1002/cpmb.56
Heng-Chang Chen, Eduard Zorita, Guillaume J. Filion
{"title":"Using Barcoded HIV Ensembles (B-HIVE) for Single Provirus Transcriptomics","authors":"Heng-Chang Chen,&nbsp;Eduard Zorita,&nbsp;Guillaume J. Filion","doi":"10.1002/cpmb.56","DOIUrl":"10.1002/cpmb.56","url":null,"abstract":"<p>The latent HIV reservoir is the main barrier to curing AIDS, because infected cells escape the immune system and antiretroviral therapies. Developing new treatment strategies requires technologies to trace latent proviruses. Here, we describe a genome-wide technique called Barcoded HIV Ensembles (B-HIVE) to measure HIV expression at the single provirus level. The principle of B-HIVE is to tag the genome of HIV with DNA barcodes to trace viral transcripts produced by single proviruses in an infected cell population. This in turn reveals which proviruses are active and which are latent or expressed at low level. B-HIVE is a high-throughput method to identify and quantify thousands of individual viral transcripts per round of infection. It can be applied in different conditions, characterizing the response of single proviruses to different treatments. Overall, B-HIVE gives unprecedented insight into the expression of single proviruses in populations of HIV-infected cells. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10734,"journal":{"name":"Current Protocols in Molecular Biology","volume":"122 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-04-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmb.56","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36179748","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 9
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