Molecular Biology

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    Protocols in Current Issue
    Plasmid and Sequencing Library Preparation for CRISPRi Barcoded Expression Reporter Sequencing (CiBER-seq) in Saccharomyces cerevisiae
    Authors:  Ryan Y Muller, Zuriah A Meacham and Nicholas T Ingolia, date: 04/05/2022, view: 683, Q&A: 0
    [Abstract]

    Genetic networks regulate nearly all biological processes, including cellular differentiation, homeostasis, and immune responses. Determining the precise role of each gene within a regulatory network can explain its overall, integrated function, and pinpoint mechanisms underlying misregulation in disease states. Transcriptional reporter assays are

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    TGIRT-seq Protocol for the Comprehensive Profiling of Coding and Non-coding RNA Biotypes in Cellular, Extracellular Vesicle, and Plasma RNAs
    Authors:  Hengyi Xu, Ryan M. Nottingham and Alan M. Lambowitz, date: 12/05/2021, view: 1182, Q&A: 0
    [Abstract]

    High-throughput RNA sequencing (RNA-seq) has extraordinarily advanced our understanding of gene expression and disease etiology, and is a powerful tool for the identification of biomarkers in a wide range of organisms. However, most RNA-seq methods rely on retroviral reverse transcriptases (RTs), enzymes that have inherently low fidelity and

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    Preparation and Characterization of Internally Modified DNA Templates for Chemical Transcription Roadblocking
    Author:  Eric J. Strobel, date: 09/05/2021, view: 1119, Q&A: 0
    [Abstract]

    Site-specific transcription arrest is the basis of emerging technologies that assess nascent RNA structure and function. Cotranscriptionally folded RNA can be displayed from an arrested RNA polymerase (RNAP) for biochemical manipulations by halting transcription elongation at a defined DNA template position. Most transcription

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    Isolation of Nuclei from Mouse Dorsal Root Ganglia for Single-nucleus Genomics
    Authors:  Lite Yang, Ivan Tochitsky, Clifford J. Woolf and William Renthal, date: 08/05/2021, view: 1861, Q&A: 0
    [Abstract]

    Primary somatosensory neurons, whose cell bodies reside in the dorsal root ganglion (DRG) and trigeminal ganglion, are specialized to transmit sensory information from the periphery to the central nervous system. Our molecular understanding of peripheral sensory neurons has been limited by both their heterogeneity and low abundance compared with

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    Transcriptional Run-on: Measuring Nascent Transcription at Specific Genomic Sites in Yeast
    Authors:  Victoria Begley, Lola de Miguel-Jiménez and Sebastián Chávez, date: 06/20/2021, view: 1528, Q&A: 0
    [Abstract]

    DNA transcription by RNA polymerases has always interested the scientific community as it is one of the most important processes involved in genome expression. This has led scientists to come up with different protocols allowing analysis of this process in specific locations across the genome by quantitating the amount of RNA polymerases

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    EmPC-seq: Accurate RNA-sequencing and Bioinformatics Platform to Map RNA Polymerases and Remove Background Error
    [Abstract]

    Transcription errors can substantially affect metabolic processes in organisms by altering the epigenome and causing misincorporations in mRNA, which is translated into aberrant mutant proteins. Moreover, within eukaryotic genomes there are specific Transcription Error-Enriched genomic Loci (TEELs) which are transcribed by RNA polymerases with

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    Analyzing (Re)Capping of mRNA Using Transcript Specific 5' End Sequencing
    Authors:  Daniel del Valle Morales and Daniel R. Schoenberg, date: 10/20/2020, view: 1913, Q&A: 0
    [Abstract] The 5′ cap is a ubiquitous feature of eukaryotic mRNAs. It is added in the nucleus onto newly synthesized pre-mRNA, and in the cytoplasm onto mRNAs after decapping or endonuclease cleavage. Cytoplasmic recapping can occur after loss of the cap at the native 5′ end, or downstream within the body of the mRNA. The identification and location of ...
    Isolation and Transcriptomic Profiling of Single Myofibers from Mice
    [Abstract] Skeletal muscle is composed of different cells and myofiber types, with distinct metabolic and structural features. Generally, transcriptomic analysis of skeletal muscle is performed using whole muscle, resulting in average information as all cells composing the organ contribute to the expression value detected for each gene with the loss of ...
    Yeast Single-cell RNA-seq, Cell by Cell and Step by Step
    [Abstract] Single-cell RNA-seq (scRNA-seq) has become an established method for uncovering the intrinsic complexity within populations. Even within seemingly homogenous populations of isogenic yeast cells, there is a high degree of heterogeneity that originates from a compact and pervasively transcribed genome. Research with microorganisms such as yeast ...
    Adapting the Smart-seq2 Protocol for Robust Single Worm RNA-seq
    [Abstract] Most nematodes are small worms that lack enough RNA for regular RNA-seq protocols without pooling hundred to thousand of individuals. We have adapted the Smart-seq2 protocol in order to sequence the transcriptome of an individual worm. While developed for individual Steinernema carpocapsae and Caenorhabditis elegans larvae as ...



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