Current Protocols in Molecular Biology最新文献

Genetic tools for specific perturbation of endogenous gene expression are highly desirable for interrogation of plant gene functions and improvement of crop traits. Synthetic transcriptional activators derived from the CRISPR/Cas9 system are emerging as powerful new tools for activating the endogenous expression of genes of interest in plants. These synthetic constructs, generated by tethering transcriptional activation domains to a nuclease-dead Cas9 (dCas9), can be directed to the promoters of endogenous target genes by single guide RNAs (sgRNAs) to activate transcription. Here, we provide a detailed protocol for targeted transcriptional activation in plants using a recently developed, highly potent dCas9 gene activator construct referred to as dCas9-TV. This protocol covers selection of sgRNA targets, construction of sgRNA expression cassettes, and screening for an optimal sgRNA using a protoplast-based promoter-luciferase assay. Finally, the dCas9-TV gene activator coupled with the optimal sgRNA is delivered into plants via Agrobacterium-mediated transformation, thereby enabling robust upregulation of target gene expression in transgenic Arabidopsis and rice plants. © 2019 by John Wiley & Sons, Inc.

Cover: Hainer and Fazzio (https://doi.org/10.1002/cpmb.85), Cartoon of workflow. Shown is a diagram of the CUT&RUN approach. See e85.

The most prevalent modified base in mRNA, N⁶-methyladenosine (m⁶A), 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 (m⁶Am), which is difficult to distinguish from m⁶A, occurs as the first transcribed nucleotide of certain transcripts. Both the m⁶A and m⁶Am modifications have been implicated in numerous biological processes, and their precise mapping is crucial to understanding their functions. To address this need, we developed miCLIP, a method that maps both m⁶A and m⁶Am at individual nucleotide resolution. Here we describe the miCLIP protocol, with slight improvements to the initially published protocol for both the experimental methodology and bioinformatics analysis. © 2019 by John Wiley & Sons, Inc.

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. In ChAR-seq, proximity ligation of RNA and DNA to a linker molecule is used to construct a chimeric RNA-DNA molecule that is converted to DNA for sequencing. In a single assay, ChAR-seq can discover de novo chromatin interactions of distinct RNAs, including nascent transcripts, splicing RNAs, and long noncoding RNAs (lncRNAs). Resulting “maps” of genome-bound RNAs should provide new insights into RNA biology. © 2019 by John Wiley & Sons, Inc.

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. Improved accuracy calibration of the instrument from the tissue surface is emphasized, as this allows for significantly improved mass determination in time of flight (TOF)-based instruments enabling more confident preliminary lipid identification. This improved initial result allows MALDI-IMS data to be complemented with additional instrumentation, such as liquid chromatography mass spectrometry workflows or specialized non-TOF systems such as Fourier transform cyclotron resonance instruments. This method is not limited to human tissue and can be applied to virtually any lipid-rich formalin-fixed tissue. © 2019 by John Wiley & Sons, Inc.

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. Therefore, CUT&RUN provides an alternative to ChIP-seq approaches for mapping chromatin proteins, which typically have relatively high signal-to-noise ratios, while using fewer cells and at a lower cost. Here, we describe the methods for performing CUT&RUN, generating DNA-sequencing libraries, and analyzing the resulting datasets. © 2019 by John Wiley & Sons, Inc.

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. © 2018 by John Wiley & Sons, Inc.

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.

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.