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Current Protocols in Molecular Biology

Magnolia Bostick, Nathalie Bolduc, Alisa Lehman, Andrew Farmer
Next-generation sequencing is empowering a deeper understanding of biology by enabling RNA expression analysis over the entire transcriptome with high sensitivity and a wide dynamic range. One powerful application within this field is stranded RNA sequencing (RNA-seq), which is necessary to distinguish overlapping genes and to conduct comprehensive annotation and quantification of long non-coding RNAs. Commonly used methods for generating strand-specific RNA-seq libraries are often complicated by protocols that require several rounds of enzymatic treatments and clean-up steps, making them time-intensive, insensitive, and unsuitable for processing several samples simultaneously...
October 10, 2016: Current Protocols in Molecular Biology
Rachel N Fish, Magnolia Bostick, Alisa Lehman, Andrew Farmer
RNA sequencing (RNA-seq) is a powerful method for analyzing cell state, with minimal bias, and has broad applications within the biological sciences. However, transcriptome analysis of seemingly homogenous cell populations may in fact overlook significant heterogeneity that can be uncovered at the single-cell level. The ultra-low amount of RNA contained in a single cell requires extraordinarily sensitive and reproducible transcriptome analysis methods. As next-generation sequencing (NGS) technologies mature, transcriptome profiling by RNA-seq is increasingly being used to decipher the molecular signature of individual cells...
October 10, 2016: Current Protocols in Molecular Biology
Nathalie Bolduc, Alisa P Lehman, Andrew Farmer
Chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing (ChIP-seq) has become the gold standard for mapping of transcription factors and histone modifications throughout the genome. However, for ChIP experiments involving few cells or targeting low-abundance transcription factors, the small amount of DNA recovered makes ligation of adapters very challenging. In this unit, we describe a ChIP-seq workflow that can be applied to small cell numbers, including a robust single-tube and ligation-free method for preparation of sequencing libraries from sub-nanogram amounts of ChIP DNA...
October 10, 2016: Current Protocols in Molecular Biology
Ana Cecilia Mestre-Citrinovitz, Adrián Jorge Sestelo, María Belén Ceballos, José Lino Barañao, Patricia Saragüeta
Cell line establishment of somatic cells is a valuable resource to preserve genetic material of rare, difficult-to-find, endangered and giant species like Jaguar (Panthera onca), the largest South American felid. This unit focuses on the isolation and culture of fibroblasts from Jaguar skin and muscle biopsies, and ear cartilage dissection immediately after death to preserve one of the several endangered species in this biome. These culture techniques enabled us to contribute 570 samples from 45 autochthonous and endangered species, including Jaguar...
October 10, 2016: Current Protocols in Molecular Biology
Shawn W Foley, Brian D Gregory
Every eukaryotic RNA transcript undergoes extensive post-transcriptional processing from the moment of transcription up through degradation. This regulation is performed by a distinct cohort of RNA-binding proteins which recognize their target transcript by both its primary sequence and secondary structure. Here, we describe protein interaction profile sequencing (PIP-seq), a technique that uses ribonuclease-based footprinting followed by high-throughput sequencing to globally assess both protein-bound RNA sequences and RNA secondary structure...
October 10, 2016: Current Protocols in Molecular Biology
Benjamin Izar, Asaf Rotem
For the last five decades, measuring the ability of cells to grow in soft agar has served as the gold standard assay for in vitro cellular transformation. Nevertheless, the soft agar colony formation assay is time consuming and ill-suited for high-throughput screens. This unit describes an equally qualitative and quantitative assay known as growth in low attachment or GILA. The GILA assay is suitable for high-throughput pharmacological or genetic screens and allows the simultaneous examination of multiple cell lines and experimental perturbations...
October 10, 2016: Current Protocols in Molecular Biology
Nan Fang, Rumeysa Akinci-Tolun
Recent advances in single-cell RNA sequencing technologies have revealed high heterogeneity of gene expression profiles in individual cells. However, most current single-cell RNA-seq methods use oligo-dT priming in the reverse transcription steps and detect only polyA-positive for more accuracy, since there are also polyA-positive non-coding RNAs transcripts, not other important RNA species, such as polyA-negative noncoding RNA. Reverse transcription using random oligos enables detection of not only the noncoding RNA species without polyA tails, but also ribosomal RNA (rRNA)...
2016: Current Protocols in Molecular Biology
Erbay Yigit, George R Feehery, Bradley W Langhorst, Fiona J Stewart, Eileen T Dimalanta, Sriharsa Pradhan, Barton Slatko, Andrew F Gardner, James McFarland, Christine Sumner, Theodore B Davis
"Microbiome" is used to describe the communities of microorganisms and their genes in a particular environment, including communities in association with a eukaryotic host or part of a host. One challenge in microbiome analysis concerns the presence of host DNA in samples. Removal of host DNA before sequencing results in greater sequence depth of the intended microbiome target population. This unit describes a novel method of microbial DNA enrichment in which methylated host DNA such as human genomic DNA is selectively bound and separated from microbial DNA before next-generation sequencing (NGS) library construction...
2016: Current Protocols in Molecular Biology
Hyun-Min Kim, Monica P Colaiácovo
The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) system is successfully being used for efficient and targeted genome editing in various organisms, including the nematode C. elegans. Recent studies have developed various CRISPR-Cas9 approaches to enhance genome engineering via two major DNA double-strand break repair pathways: non-homologous end joining and homologous recombination. Here we describe a protocol for Cas9-mediated C. elegans genome editing together with single guide RNA (sgRNA) and repair template cloning, as well as injection methods required for delivering Cas9, sgRNAs, and repair template DNA into the C...
2016: Current Protocols in Molecular Biology
Xingliang Ma, Yao-Guang Liu
The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated genome targeting system has been applied to a variety of organisms, including plants. Compared to other genome-targeting technologies such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), the CRISPR/Cas9 system is easier to use and has much higher editing efficiency. In addition, multiple "single guide RNAs" (sgRNAs) with different target sequences can be designed to direct the Cas9 protein to multiple genomic sites for simultaneous multiplex editing...
2016: Current Protocols in Molecular Biology
Joel R McDade, Nicole C Waxmonsky, Lianna E Swanson, Melina Fan
CRISPR/Cas9 technology is ideally suited for genome-wide screening applications due to the ease of generating guide RNAs (gRNAs) and the versatility of Cas9 or Cas9 derivatives to knockout, repress, or activate expression of target genes. Several pooled lentiviral CRISPR libraries have been developed and are now publicly available, but while using CRISPR/Cas9 for genetic experiments has become widely adopted, genome-wide screening experiments remain technically challenging. This review covers the basics of CRISPR/Cas9, describes several publicly available CRISPR libraries, and provides a general protocol for conducting genome-wide screening experiments using CRISPR/Cas9...
2016: Current Protocols in Molecular Biology
Martin Newman, Frederick M Ausubel
Until very recently, the prospect of introducing mutations or exogenous DNA sequences at precise locations in the genomes of plants and animals was difficult, if not impossible. This rapidly changed with the demonstration that the type II CRISPR-Cas complex, a bacterial anti-viral surveillance system, could be engineered into a simple and robust platform for introducing double-stranded DNA breaks at nearly any position of plant and animal genomes. The prospect of efficiently creating tailored changes to a gene of interest is revolutionizing biomedical research, allowing exciting new questions to be asked...
2016: Current Protocols in Molecular Biology
Hiroko Wakimoto, J G Seidman, Roger S Y Foo, Jianming Jiang
RNA interference (RNAi) is a rapid approach to dissect loss-of-function phenotype for a gene of interest. However, it is challenging to perform RNAi in specific organs and tissues in vivo. Engineered viruses can provide a useful tool for delivery of small RNAs in vivo. Recombinant adeno-associated viruses (rAAVs) are the preferred method for delivering genes or gene modulators to target cells due to their high titer, low immune response, ability to transduce many types of cell, and overall safety. In this unit, we describe protocols for use of rAAVs as a cargo to deliver miRNA backbone-based shRNA controlled by a cardiac-specific promoter into the mouse heart...
2016: Current Protocols in Molecular Biology
Seda Eminaga, Polakit Teekakirikul, Christine E Seidman, Jonathan G Seidman
This unit describes a step-by-step protocol to detect and quantify proliferating cells in paraffin-embedded tissue sections. Two well-established markers of proliferation (incorporation of BrdU into newly synthesized DNA and expression of the nuclear protein Ki67) are detected after antigen-retrieval and subsequent immunofluorescence staining and confocal microscopy. © 2016 by John Wiley & Sons, Inc.
2016: Current Protocols in Molecular Biology
Bryan Sands, Roger Brent
In 1973, Cohen and coworkers published a foundational paper describing the cloning of DNA fragments into plasmid vectors. In it, they used DNA segments made by digestion with restriction enzymes and joined these in vitro with DNA ligase. These methods established working recombinant DNA technology and enabled the immediate start of the biotechnology industry. Since then, "classical" recombinant DNA technology using restriction enzymes and DNA ligase has matured. At the same time, researchers have developed numerous ways to generate large, complex, multisegment DNA constructions that offer advantages over classical techniques...
January 2016: Current Protocols in Molecular Biology
Tineke L Lenstra, Daniel R Larson
Visualization of single RNA molecules in living cells has enabled the study of synthesis, movement, and localization of mRNAs and has provided insight into gene regulation with sub-second temporal resolution and nanometer spatial resolution. Following transcription in single cells indicates that gene activity is heterogeneous between cells and also exhibits random variability over time even within single cells. Studies of mRNAs in yeast can take advantage of the powerful genetics available in this model organism and allow mechanistic questions to be addressed...
2016: Current Protocols in Molecular Biology
Nan Fang, Dirk Löffert, Rumeysa Akinci-Tolun, Katja Heitz, Alexander Wolf
Circulating DNA is cell-free DNA (cfDNA) in serum or plasma that can be used for non-invasive prenatal testing, as well as cancer diagnosis, prognosis, and stratification. High-throughput sequence analysis of the cfDNA with next-generation sequencing technologies has proven to be a highly sensitive and specific method in detecting and characterizing mutations in cancer and other diseases, as well as aneuploidy during pregnancy. This unit describes detailed procedures to extract circulating cfDNA from human serum and plasma and generate sequencing libraries from a wide concentration range of circulating DNA...
2016: Current Protocols in Molecular Biology
Oliver Fiehn
Gas chromatography-mass spectrometry (GC-MS)-based metabolomics is ideal for identifying and quantitating small-molecule metabolites (<650 Da), including small acids, alcohols, hydroxyl acids, amino acids, sugars, fatty acids, sterols, catecholamines, drugs, and toxins, often using chemical derivatization to make these compounds sufficiently volatile for gas chromatography. This unit shows how GC-MS-based metabolomics allows integration of targeted assays for absolute quantification of specific metabolites with untargeted metabolomics to discover novel compounds...
2016: Current Protocols in Molecular Biology
Ozlem Yildirim, Robert E Kingston
DNA synthesis and chromatin assembly are the two most critical processes of eukaryotic cell division. It is well known that their coordination is tightly regulated. Although the interplay between DNA and its higher-order chromatin state is integral for many processes, including cell survival and genome stability, little is known about the re-establishment of chromatin structure during the cell cycle. Moreover, the extent to which the fidelity of the newly synthesized chromatin plays a role in the maintenance of cellular identity is still under debate...
2016: Current Protocols in Molecular Biology
Chao-Kai Chou, Pei-Hsiang Tsou, Jennifer L Hsu, Heng-Huan Lee, Ying-Nai Wang, Jun Kameoka, Mien-Chie Hung
Signal transduction is essential for maintaining normal cell physiological functions, and deregulation of signaling can lead to diseases such as diabetes and cancers. Some of the major players in signal delivery are molecular complexes composed of proteins and nucleic acids. This unit describes a technique called microchannel for multiparameter analysis of proteins in a single complex (mMAPS) for analyzing and quantifying individual target signaling complexes. mMAPS is a flow-proteometric system that allows detection of individual proteins or complexes flowing through a microfluidic channel...
2016: Current Protocols in Molecular Biology
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