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    Generation of microRNA Sponge Library
    Author:  Sebastian Herzog, date: 04/20/2018, view: 48, Q&A: 0
    [Abstract] This protocol describes the generation and functional validation of microRNA (miRNA) sponge or decoy constructs. When expressed from a strong promoter, these transcripts can sequester specific miRNA:RISC complexes, thereby resulting in a derepression of endogenous target mRNA. Hence, cells expressing such sponges display a partial or full miRNA ...
    RNA Purification from the Unicellular Green Alga, Chromochloris zofingiensis
    Authors:  Sean D. Gallaher and Melissa S. Roth, date: 04/05/2018, view: 164, Q&A: 0
    [Abstract] Chromochloris zofingiensis is a unicellular green alga that is an emerging model species for studies in fields such as biofuel production, ketocarotenoid biosynthesis and metabolism. The recent availability of a high-quality genome assembly facilitates systems-level analysis, such as RNA-Seq. However, cells of this alga have a tough cell ...
    Isolation of Nuclei in Tagged Cell Types (INTACT), RNA Extraction and Ribosomal RNA Degradation to Prepare Material for RNA-Seq
    [Abstract] Gene expression is dynamically regulated on many levels, including chromatin accessibility and transcription. In order to study these nuclear regulatory events, we describe our method to purify nuclei with Isolation of Nuclei in TAgged Cell Types (INTACT). As nuclear RNA is low in polyadenylated transcripts and conventional pulldown methods would ...
    Detection and Analysis of Circular RNAs by RT-PCR
    Authors:  Amaresh C Panda and Myriam Gorospe, date: 03/20/2018, view: 833, Q&A: 0
    [Abstract] Gene expression in eukaryotic cells is tightly regulated at the transcriptional and posttranscriptional levels. Posttranscriptional processes, including pre-mRNA splicing, mRNA export, mRNA turnover, and mRNA translation, are controlled by RNA-binding proteins (RBPs) and noncoding (nc)RNAs. The vast family of ncRNAs comprises diverse regulatory ...
    RNA Cap Methyltransferase Activity Assay
    Authors:  Jackson B. Trotman and Daniel R. Schoenberg, date: 03/20/2018, view: 335, Q&A: 0
    [Abstract] Methyltransferases that methylate the guanine-N7 position of the mRNA 5’ cap structure are ubiquitous among eukaryotes and commonly encoded by viruses. Here we provide a detailed protocol for the biochemical analysis of RNA cap methyltransferase activity of biological samples. This assay involves incubation of cap-methyltransferase-containing ...
    Visualization of RNA 3’ ends in Escherichia coli Using 3’ RACE Combined with Primer Extension
    Authors:  Xun Wang, Heung Jin Jeon, Monford Paul Abishek N, Jin He and Heon M. Lim, date: 03/05/2018, view: 356, Q&A: 0
    [Abstract] In this assay, 3’ RACE (Rapid Amplification of cDNA 3’ Ends) followed by PE (primer extension), abbreviated as 3’ RACE-PE is used to identify the mRNA 3’ ends. The following protocol describes the amplification of the mRNA 3’ ends at the galactose operon in E. coli and the corresponding visualization of the PCR products through PE. In PE, ...
    Construction and Cloning of Minigenes for in vivo Analysis of Potential Splice Mutations
    Authors:  Lisa Maria Riedmayr, Sybille Böhm, Stylianos Michalakis and Elvir Becirovic, date: 03/05/2018, view: 330, Q&A: 0
    [Abstract] Disease-associated mutations influencing mRNA splicing are referred to as splice mutations. The majority of splice mutations are found on exon-intron boundaries defining canonical donor and acceptor splice sites. However, mutations in the coding region (exonic mutations) can also affect mRNA splicing. Exact knowledge of the disease mechanism of ...
    Terminal Deoxynucleotidyl Transferase Mediated Production of Labeled Probes for Single-molecule FISH or RNA Capture
    Authors:  Imre Gaspar, Frank Wippich and Anne Ephrussi, date: 03/05/2018, view: 435, Q&A: 0
    [Abstract] Arrays of short, singly-labeled ssDNA oligonucleotides enable in situ hybridization with single molecule sensitivity and efficient transcript specific RNA capture. Here, we describe a simple, enzymatic protocol that can be carried out using basic laboratory equipment to convert arrays of PCR oligos into smFISH and RAP probesets in a ...
    A Small RNA Isolation and Sequencing Protocol and Its Application to Assay CRISPR RNA Biogenesis in Bacteria
    [Abstract] Next generation high-throughput sequencing has enabled sensitive and unambiguous analysis of RNA populations in cells. Here, we describe a method for isolation and strand-specific sequencing of small RNA pools from bacteria that can be multiplexed to accommodate multiple biological samples in a single experiment. Small RNAs are isolated by ...
    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 ...