Current Protocols in Molecular Biology最新文献_第4页

Auxin-Inducible Degron System for Depletion of Proteins in Saccharomyces cerevisiae 酿酒酵母蛋白质耗竭的生长素诱导脱菌系统

Current Protocols in Molecular Biology Pub Date : 2019-09-06 DOI: 10.1002/cpmb.104

Ameet Shetty, Natalia I. Reim, Fred Winston

引用次数: 10

Whole-Genome Sequencing of Yeast Cells 酵母细胞全基因组测序

Current Protocols in Molecular Biology Pub Date : 2019-09-05 DOI: 10.1002/cpmb.103

Rajaraman Gopalakrishnan, Fred Winston

{"title":"Whole-Genome Sequencing of Yeast Cells","authors":"Rajaraman Gopalakrishnan, Fred Winston","doi":"10.1002/cpmb.103","DOIUrl":"10.1002/cpmb.103","url":null,"abstract":"The budding yeast, Saccharomyces cerevisiae, has been widely used for genetic studies of fundamental cellular functions. The isolation and analysis of yeast mutants is a commonly used and powerful technique to identify the genes that are involved in a process of interest. Furthermore, natural genetic variation among wild yeast strains has been studied for analysis of polygenic traits by quantitative trait loci mapping. Whole-genome sequencing, often combined with bulk segregant analysis, is a powerful technique that helps determine the identity of mutations causing a phenotype. Here, we describe protocols for the construction of libraries for S. cerevisiae whole-genome sequencing. We also present a bioinformatic pipeline to determine the genetic variants in a yeast strain using whole-genome sequencing data. This pipeline can also be used for analyzing Schizosaccharomyces pombe mutants. © 2019 by John Wiley & Sons, Inc.Basic Protocol 1: Generation of haploid spores for bulk segregant analysisBasic Protocol 2: Extraction of genomic DNA from yeast cellsBasic Protocol 3: Shearing of genomic DNA for library preparationBasic Protocol 4: Construction and amplification of DNA librariesSupport Protocol 1: Annealing oligonucleotides for forming Y-adaptersSupport Protocol 2: Size selection and cleanup using SPRI beadsBasic Protocol 5: Identification of genomic variants from sequencing data","PeriodicalId":10734,"journal":{"name":"Current Protocols in Molecular Biology","volume":"128 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmb.103","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"81353136","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: 12

Probing In Vivo Structure of Individual mRNA 3′ Isoforms Using Dimethyl Sulfate 用硫酸二甲酯探测单个mRNA 3 '异构体的体内结构

Current Protocols in Molecular Biology Pub Date : 2019-07-31 DOI: 10.1002/cpmb.99

Zarmik Moqtaderi, Joseph V. Geisberg

{"title":"Probing In Vivo Structure of Individual mRNA 3′ Isoforms Using Dimethyl Sulfate","authors":"Zarmik Moqtaderi, Joseph V. Geisberg","doi":"10.1002/cpmb.99","DOIUrl":"10.1002/cpmb.99","url":null,"abstract":"The DMS region extraction and deep sequencing (DREADS) procedure was designed to probe RNA structure in vivo and to link this structural information to specific 3′ isoforms. Growing cells are treated with the alkylating agent dimethyl sulfate (DMS), which enters easily into cells and modifies RNA molecules at solvent-exposed A and C residues. RNA is isolated, and sequencing libraries are constructed in a manner that preserves the identities of individual mRNA isoforms arising from alternative cleavage/polyadenylation sites. During the cDNA synthesis step of library construction, the progress of reverse transcriptase (RT) is blocked when it encounters a DMS modification on the RNA, leading to disproportionate cDNA termination adjacent to DMS-modified positions. After paired-end deep sequencing, the downstream end of each sequenced fragment is mapped to a specific cleavage/poly(A) site representing an individual mRNA 3′ isoform. The upstream mapped end of the sequenced fragment defines where the RT reaction stopped. Over the population of all sequenced fragments derived from a particular isoform, A and C positions that are overrepresented next to the upstream endpoints in the DMS sample (relative to a parallel untreated control) are inferred to have been DMS modified, and hence solvent exposed. This method thus allows in vivo structural information obtained using DMS to be linked to individual mRNA 3′ isoforms. © 2019 by John Wiley & Sons, Inc.","PeriodicalId":10734,"journal":{"name":"Current Protocols in Molecular Biology","volume":"128 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-07-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmb.99","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41194054","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

Protein Binding to mRNA 3′ Isoforms 蛋白质与mRNA 3 '亚型的结合

Current Protocols in Molecular Biology Pub Date : 2019-07-31 DOI: 10.1002/cpmb.101

Joseph V. Geisberg, Zarmik Moqtaderi

引用次数: 1

Generating Single Cell–Derived Knockout Clones in Mammalian Cells with CRISPR/Cas9 利用CRISPR/Cas9在哺乳动物细胞中产生单细胞来源的敲除克隆

Current Protocols in Molecular Biology Pub Date : 2019-07-26 DOI: 10.1002/cpmb.100

Christopher J. Giuliano, Ann Lin, Vishruth Girish, Jason M. Sheltzer

引用次数: 65

Issue Information TOC 发布信息TOC

Current Protocols in Molecular Biology Pub Date : 2019-06-19 DOI: 10.1002/cpmb.76

引用次数: 0

In Situ Hybridization for Detecting Mature MicroRNAs In Vivo at Single-Cell Resolution 原位杂交技术在单细胞分辨率下检测体内成熟的microrna

Current Protocols in Molecular Biology Pub Date : 2019-06-04 DOI: 10.1002/cpmb.93

Amanda L. Minogue, Swathi Arur

引用次数: 0

Single-Cell RNA-seq: Introduction to Bioinformatics Analysis 单细胞RNA-seq:生物信息学分析导论

Current Protocols in Molecular Biology Pub Date : 2019-05-24 DOI: 10.1002/cpmb.92

Fei Ji, Ruslan I. Sadreyev

引用次数: 8

Digital Droplet PCR for Monitoring Tissue-Specific Cell Death Using DNA Methylation Patterns of Circulating Cell-Free DNA 利用循环游离细胞DNA的DNA甲基化模式监测组织特异性细胞死亡的数字液滴PCR

Current Protocols in Molecular Biology Pub Date : 2019-04-23 DOI: 10.1002/cpmb.90

Ruth Shemer, Judith Magenheim, Yuval Dor

{"title":"Digital Droplet PCR for Monitoring Tissue-Specific Cell Death Using DNA Methylation Patterns of Circulating Cell-Free DNA","authors":"Ruth Shemer, Judith Magenheim, Yuval Dor","doi":"10.1002/cpmb.90","DOIUrl":"10.1002/cpmb.90","url":null,"abstract":"Cell death involves the release of short DNA fragments into blood, termed circulating cell-free DNA (cfDNA). Sequencing of cfDNA in the plasma has recently emerged as a liquid biopsy for detecting fetal chromosomal aberrations, tumor DNA, and graft rejection. However, in cases where cfDNA is derived from tissues with a normal genome, its primary sequence is not informative regarding the tissue of origin. We developed a method of determining the tissue origins of cfDNA, allowing inference of tissue-specific cell death, based on tissue-specific methylation patterns. We have previously described a version of the method that uses next generation sequencing (NGS) to determine methylation patterns in specific marker loci. Here we describe a rapid and simple procedure for cfDNA methylation analysis using droplet digital PCR (ddPCR) on bisulfite treated cfDNA to accurately count the number of molecules carrying a specific methylation signature. Specificity and sensitivity of the assay increases by simultaneously interrogating four to six cytosines in the same molecule using two fluorescent probes. cfDNA methylation analysis using ddPCR can find multiple applications in the non-invasive study of human tissue dynamics in health and disease. © 2019 by John Wiley & Sons, Inc.","PeriodicalId":10734,"journal":{"name":"Current Protocols in Molecular Biology","volume":"127 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-04-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmb.90","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37363386","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

Low-Input MNase Accessibility of Chromatin (Low-Input MACC) 染色质低输入mnc可及性(低输入MACC)

Current Protocols in Molecular Biology Pub Date : 2019-04-06 DOI: 10.1002/cpmb.91

Mattia Lion, Michael Y. Tolstorukov, Marjorie A. Oettinger

引用次数: 1