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Current Protocols in Molecular Biology

Ben R Hawley, Samie R Jaffrey
The most prevalent modified base in mRNA, N6 -methyladenosine (m6 A), is found in several thousand transcripts, typically near the stop codon, although it can occur anywhere in the mRNA. In addition, the highly similar nucleotide N6 ,2'-O-dimethyladenosine (m6 Am), which is difficult to distinguish from m6 A, occurs as the first transcribed nucleotide of certain transcripts. Both the m6 A and m6 Am modifications have been implicated in numerous biological processes, and their precise mapping is crucial to understanding their functions...
March 15, 2019: Current Protocols in Molecular Biology
David Jukam, Charles Limouse, Owen K Smith, Viviana I Risca, Jason C Bell, Aaron F Straight
RNA is a fundamental component of chromatin. Noncoding RNAs (ncRNAs) can associate with chromatin to influence gene expression and chromatin state; many also act at long distances from their transcriptional origin. Yet we know almost nothing about the functions or sites of action for most ncRNAs. Current methods to identify sites of RNA interaction with the genome are limited to the study of a single RNA at a time. Here we describe a protocol for ChAR-seq, a strategy to identify all chromatin-associated RNAs and map their DNA contacts genome-wide...
February 20, 2019: Current Protocols in Molecular Biology
Matthew B O'Rourke, Caine C Smith, Suzanne M De La Monte, Greg T Sutherland, Matthew P Padula
Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) is a well-established technique for elucidating the location and relative abundance of a range of biomolecules. More recently, research into this technique has shifted from simple discovery and demonstration of utility to application in biomedical research. Here, we describe a protocol utilizing MALDI-IMS for the spatial mapping of lipids in brain tissue from normal human brains and brains from patients with Alzheimer's disease, in the context of Alzheimer's disease...
February 8, 2019: Current Protocols in Molecular Biology
Sarah J Hainer, Thomas G Fazzio
Determining the genomic location of DNA-binding proteins is essential to understanding their function. Cleavage Under Targets and Release Using Nuclease (CUT&RUN) is a powerful method for mapping protein-DNA interactions at high resolution. In CUT&RUN, a recombinant protein A-microccocal nuclease (pA-MN) fusion is recruited by an antibody targeting the chromatin protein of interest; this can be done with either uncrosslinked or formaldehyde-crosslinked cells. DNA fragments near sites of antibody binding are released from the insoluble bulk chromatin through endonucleolytic cleavage and used to build barcoded DNA-sequencing libraries that can be sequenced in pools of at least 30...
January 28, 2019: Current Protocols in Molecular Biology
Anna B Seminara, Asan Turdiev, Husan Turdiev, Vincent T Lee
Protein interactions with nucleic acids are important for the synthesis, regulation, and stability of macromolecules. While a number of assays are available for interrogating these interactions, the differential radial capillary action of ligand assay (DRaCALA) has been developed as an easy and flexible platform that allows for the study of individual interactions when carrying out high-throughput screening for novel binding proteins and small molecule inhibitors. In this article, we describe the principle of DRaCALA and methods that utilize DRaCALA to determine the affinity and specificity of individual protein-nucleic acid interactions as well as uses for screening for binding proteins and chemical inhibitors...
December 3, 2018: Current Protocols in Molecular Biology
Karen L Elbing, Roger Brent
We provide protocols for titering and isolating bacterial colonies from single cells by serial dilutions, for streaking agar plates, and for spreading suspensions of cells on plates. Support protocols describe replica plating and methods for storing strains as agar stabs and frozen stocks. © 2018 by John Wiley & Sons, Inc.
November 10, 2018: Current Protocols in Molecular Biology
Karen L Elbing, Roger Brent
We describe the procedure for inoculating overnight (starter) cultures of E. coli from a single colony, along with considerations for growing larger cultures. We also include two methods for monitoring the number of cells per unit volume (density) of liquid cultures using a spectrophotometer and a hemacytometer or "count slide." © 2018 by John Wiley & Sons, Inc.
November 9, 2018: Current Protocols in Molecular Biology
Karen L Elbing, Roger Brent
In this article, we provide information about culture media, including minimal liquid media, rich liquid media, solid media, top agar, and stab agar. We also provide descriptions and useful information about tools used with growth media such as inoculating loops, sterile toothpicks, and spreaders. © 2018 by John Wiley & Sons, Inc.
November 9, 2018: Current Protocols in Molecular Biology
Maria Rita Sciuto, Valeria Coppola, Gioacchin Iannolo, Ruggero De Maria, Tobias L Haas
In the past few decades, numerous approaches have been developed to investigate protein-protein and protein-nucleic acid interactions (PPIs and PNIs). Affinity purification methods such as co-immunoprecipitation (co-IP) are commonly used to detect and isolate the macromolecular complexesresulting from these interactions. In this article, we describe a two-step co-immunoprecipitation (TIP) technique. As compared to standard co-IP, TIP provides increased specificity in the isolation of PPIs or PNIs under native expression conditions, dramatically reducing the abundance of nonspecific binders and thus facilitating downstream analyses of the interaction complexes...
October 30, 2018: Current Protocols in Molecular Biology
Mark E Lee, Xiaoyi Dou, Yingmin Zhu, Kevin J Phillips
Differential Scanning Fluorimetry Guided Refolding (DGR) is a simple methodology that can be used to rapidly screen for and identify conditions capable of accurately refolding protein preparations, such as those obtained from Escherichia coli inclusion bodies. It allows for the production in E. coli of functional proteins that would otherwise require far more expensive production methods. This unit describes how to set up a DGR refolding assay, perform DGR refolding trials in microplate format, use MeltTraceur Web software to interactively analyze the resulting data, scale-up protein production via refolding, and lastly, validate that the protein is properly folded...
October 29, 2018: Current Protocols in Molecular Biology
Eric S Pringle, Craig McCormick, Zhenyu Cheng
Post-transcriptional regulation is an important aspect of the control of gene expression. mRNAs are translated with variable efficiencies, and these efficiencies can change rapidly during adaptation to diverse environmental factors, including cellular stresses and microbial infections. Polysome profiling analysis utilizes ultracentrifugation to isolate complexes of mRNAs in the process of translation and corresponding proteins on the basis of density. Here we describe polysome profiling analysis using a continuous ultraviolet spectrophotometer and a gradient fractionator...
October 29, 2018: Current Protocols in Molecular Biology
Ben B Li, Changli Qian, Thomas M Roberts, Jean J Zhao
This unit describes a reverse transcription-quantitative PCR (RT-qPCR)-based method for gene-targeted measurement of RNA translation levels. The method includes washing and lysing cells with a buffer containing cycloheximide to enrich ribosomal accumulation at translation initiation sites (TIS), followed by enzymatic treatment to generate ribosomal footprints, reverse transcription targeted towards TIS of specific transcripts of interest to generate complementary DNA (cDNA), and qPCR to measure the abundance of these footprints...
October 22, 2018: Current Protocols in Molecular Biology
Murat Cetinbas, Shen Yu, Ruslan I Sadreyev
Identification of spontaneous or chemically induced bacterial mutations is a powerful tool for investigation of molecular mechanisms, including the mechanism of action of novel antibiotics. However, a major bottleneck to this approach has been the identification of the causative mutation underlying a phenotype of interest. Until recently, this has required time-consuming genetic analysis. However, the advent of relatively inexpensive and rapid next-generation sequencing (NGS) technologies has revolutionized the correlation of bacterial phenotypes and genotypes...
October 2018: Current Protocols in Molecular Biology
Fei Ji, Ruslan I Sadreyev
Quantitative analysis of gene expression is crucial for understanding the molecular mechanisms underlying genome regulation. RNA-seq is a powerful platform for comprehensive investigation of the transcriptome. In this unit, we present a general bioinformatics workflow for the quantitative analysis of RNA-seq data and describe a few current publicly available computational tools applicable at various steps of this workflow. These tools comprise a pipeline for quality assessment and quantitation of RNA-seq data that starts from raw sequencing files and is focused on the identification and analysis of genes that are differentially expressed between biological conditions...
October 2018: Current Protocols in Molecular Biology
Sakthimala Jagadeesan, Abdul Hakkim
RNAi is a powerful reverse genetics tool that has revolutionized genetic studies in model organisms. The bacteriovorous nematode Caenorhabditis elegans can be genetically manipulated by feeding it an Escherichia coli strain that expresses a double-stranded RNA (dsRNA) corresponding to a C. elegans gene, which leads to systemic silencing of the gene. This unit describes protocols for performing an automated high-throughput RNAi screen utilizing a full-genome C. elegans RNAi library. The protocols employ liquid-handling robotics and 96-well plates...
October 2018: Current Protocols in Molecular Biology
Sakthimala Jagadeesan, Abdul Hakkim
Automated or semi-automated high-throughput RNAi screens are highly prone to systematic errors because of multistep repetitive protocols and extensive use of automated instruments. A well-designed RNAi library can help detect and minimize systematic human and robotic errors. In this unit, we describe how to design an RNAi bacterial library for use in conjunction with the well-studied nematode Caenorhabditis elegans for automated phenotypic screens. We provide strategies to design and assemble RNAi libraries to reduce or eliminate systematic errors...
October 2018: Current Protocols in Molecular Biology
Zhe Ji
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...
October 2018: Current Protocols in Molecular Biology
Zhe Ji
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...
October 2018: Current Protocols in Molecular Biology
F William Studier
Inducible T7 expression systems are capable of producing a wide range of proteins in E. coli. 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...
October 2018: Current Protocols in Molecular Biology
Cheryl Isaac Murphy, Helen Piwnica-Worms, Stefan Grünwald, William G Romanow, Nicole Francis, Hua-Ying Fan, Sharon Marr
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 & Sons, Inc.
July 2018: Current Protocols in Molecular Biology
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