Springer protocols feed by Microbiology 
This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.
This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Mapping the Transcriptome-Wide Landscape of RBP Binding Sites Using gPAR-CLIP-seq: Bioinformatic Analysis
Protein–RNA interactions are integral components of posttranscriptional gene regulatory processes including mRNA processing and assembly of cellular architectures. Dysregulation of RNA-binding protein (RBP) expression or disruptions in RBP–RNA interactions underlie a variety of human pathologies and genetic diseases including cancer and neurodegenerative diseases (reviewed in (Cooper et al., Cell 136(4):777–793, 2009; Darnell, Cancer Res Treat 42(3):125–129, 2010; Lukong et al., Trends Genet 24 (8):416–425, 2008)). Recent studies have uncovered only a small proportion of the extensive RBP&ndas...
Source: Springer protocols feed by Microbiology - October 21, 2015 Category: Microbiology Source Type: news
Mapping the Transcriptome-Wide Landscape of RBP Binding Sites Using gPAR-CLIP-seq: Experimental Procedures
An estimated 5–10 % of protein-coding genes in eukaryotic genomes encode RNA-binding proteins (RBPs). Through dynamic changes in RNA recognition, RBPs posttranscriptionally regulate the biogenesis, transport, inheritance, storage, and degradation of RNAs. Understanding such widespread RBP-mediated posttranscriptional regulatory mechanisms requires comprehensive discovery of the in vivo binding sites of RBPs. Here, we describe the experimental procedures of the gPAR-CLIP-seq (global photoactivatable-ribonucleoside-enhanced cross-linking and precipitation followed by deep sequencing) approach we recently developed for ...
Source: Springer protocols feed by Microbiology - October 21, 2015 Category: Microbiology Source Type: news
Comparative Transcriptomics in Yeasts
Comparative functional genomics approaches have already shed an important light on the evolution of gene expression that underlies phenotypic diversity. However, comparison across many species in a phylogeny presents several major challenges. Here, we describe our experimental framework for comparative transcriptomics in a complex phylogeny. (Source: Springer protocols feed by Microbiology)
Source: Springer protocols feed by Microbiology - October 21, 2015 Category: Microbiology Source Type: news
Pathogen Gene Expression Profiling During Infection Using a Nanostring nCounter Platform
NanoString nCounter is a recently developed platform that can make direct multiplexed measurement of gene expression using color-coded probe pairs (Geiss et al., Nat Biotechnol 26(3):317–325, 2008; Malkov et al., BMC Res Notes 2:80, 2009). We have found that this platform is uniquely suitable for quantification of pathogen gene expression during infection, where pathogen RNA comprises a tiny portion of total RNA isolated from the infected tissue. Here, we describe a protocol that we have successfully applied to a number of pathogens across multiple infection models, including both invasive and mucosal infection by Ca...
Source: Springer protocols feed by Microbiology - October 21, 2015 Category: Microbiology Source Type: news
Enhancing Structural Annotation of Yeast Genomes with RNA-Seq Data
We describe a computational approach to enhance structural annotation of yeast genomes based on RNA-Seq data exploitation. The proposed pipeline is primarily based on read mapping with TopHat2. Mapping outputs are then used for various applications such as: (1) validation of exon–exon junctions of predicted transcripts, (2) definition of new transcribed features, (3) prediction of 3' UTR, and (4) identification of extra features absent from the genome assembly. We strongly encourage curators to proceed to a manual validation and editing of the reference genome. Releasing genomes with high-quality annotation is an imp...
Source: Springer protocols feed by Microbiology - October 21, 2015 Category: Microbiology Source Type: news
Predicting Gene and Genomic Regulation in Saccharomyces cerevisiae, using the YEASTRACT Database: A Step-by-Step Guided Analysis
Transcriptional regulation is one of the key steps in the control of gene expression, with huge impact on the survival, adaptation, and fitness of all organisms. However, it is becoming increasingly clear that transcriptional regulation is far more complex than initially foreseen. In model organisms such as the yeast Saccharomyces cerevisiae evidence has been piling up showing that the expression of each gene can be controlled by several transcription factors, in the close dependency of the environmental conditions. Furthermore, transcription factors work in intricate networks, being themselves regulated at the transcripti...
Source: Springer protocols feed by Microbiology - October 21, 2015 Category: Microbiology Source Type: news
Reconstruction and Analysis of the Evolution of Modular Transcriptional Regulatory Programs Using Arboretum
Comparative functional genomics aims to measure and compare genome-wide functional data such as transcriptomes, proteomes, and epigenomes across multiple species to study the conservation and divergence patterns of such quantitative measurements. However, computational methods to systematically compare these quantitative genomic profiles across multiple species are in their infancy. We developed Arboretum, a novel algorithm to identify modules of co-expressed genes and trace their evolutionary history across multiple species from a complex phylogeny. To interpret the results from Arboretum we developed several measures to ...
Source: Springer protocols feed by Microbiology - October 21, 2015 Category: Microbiology Source Type: news
Experimental Evolution and Resequencing Analysis of Yeast
Experimental evolution of microbes is a powerful tool to study adaptation to strong selection, the mechanism of evolution and the development of new traits. The development of high-throughput sequencing methods has given researchers a new ability to cheaply and easily identify mutations genome wide that are selected during the course of experimental evolution. Here we provide a protocol for conducting experimental evolution of yeast using chemostats, including fitness measurement and whole genome sequencing of evolved clones or populations collected during the experiment. Depending on the number of generations appropriate ...
Source: Springer protocols feed by Microbiology - October 21, 2015 Category: Microbiology Source Type: news
Using RNA-seq for Analysis of Differential Gene Expression in Fungal Species
The ability to extract, identify and annotate large amounts of biological data is a key feature of the “omics” era, and has led to an explosion in the amount of data available. One pivotal advance is the use of Next-Generation Sequencing (NGS) techniques such as RNA-sequencing (RNA-seq). RNA-seq uses data from millions of small mRNA transcripts or “reads” which are aligned to a reference genome. Comparative transcriptomics analyses using RNA-seq can provide the researcher with a comprehensive view of the cells’ response to a given environment or stimulus. (Source: Springer protocols feed by Microbiology)
Source: Springer protocols feed by Microbiology - October 21, 2015 Category: Microbiology Source Type: news
On the Mapping of Epistatic Genetic Interactions in Natural Isolates: Combining Classical Genetics and Genomics
We present here a general workflow to identify epistatic interactions between independently evolving loci in natural populations of the yeast Saccharomyces cerevisiae. The idea is to exploit the genetic diversity present in the species by evaluating a large number of crosses and analyzing the phenotypic distribution in the offspring. For a cross of interest, both parental strains would have a similar phenotypic value, whereas the resulting offspring would have a bimodal distribution of the phenotype, possibly indicating the presence of epistasis. Classical segregation analysis of the tetrads uncovers the penetrance and com...
Source: Springer protocols feed by Microbiology - October 21, 2015 Category: Microbiology Source Type: news
Identification of Links Between Cellular Pathways by Genetic Interaction Mapping (GIM)
We describe here protocols for preparing the pools of haploid double mutant S. cerevisiae cells, testing their composition with barcode microarrays, and analyzing the results to extract useful functional information. (Source: Springer protocols feed by Microbiology)
Source: Springer protocols feed by Microbiology - October 21, 2015 Category: Microbiology Source Type: news
Profiling of Yeast Lipids by Shotgun Lipidomics
Lipidomics is a rapidly growing technology for identification and quantification of a variety of cellular lipid molecules. Following the successful development and application of functional genomic technologies in yeast Saccharomyces cerevisiae, we witness a recent expansion of lipidomics applications in this model organism. The applications include detailed characterization of the yeast lipidome as well as screening for perturbed lipid phenotypes across hundreds of yeast gene deletion mutants. In this chapter, we describe sample handling, mass spectrometry, and bioinformatics methods developed for yeast lipidomics studies...
Source: Springer protocols feed by Microbiology - October 21, 2015 Category: Microbiology Source Type: news
Label-Free Quantitative Proteomics in Yeast
Label-free bottom-up shotgun MS-based proteomics is an extremely powerful and simple tool to provide high quality quantitative analyses of the yeast proteome with only microgram amounts of total protein. Although the experimental design of this approach is rather straightforward and does not require the modification of growth conditions, proteins or peptides, several factors must be taken into account to benefit fully from the power of this method. Key factors include the choice of an appropriate method for the preparation of protein extracts, careful evaluation of the instrument design and available analytical capabilitie...
Source: Springer protocols feed by Microbiology - October 21, 2015 Category: Microbiology Source Type: news
Single-Step Affinity Purification (ssAP) and Mass Spectrometry of Macromolecular Complexes in the Yeast S. cerevisiae
Cellular functions are mostly defined by the dynamic interactions of proteins within macromolecular networks. Deciphering the composition of macromolecular complexes and their dynamic rearrangements is the key to getting a comprehensive picture of cellular behavior and to understanding biological systems. In the last decade, affinity purification coupled to mass spectrometry has emerged as a powerful tool to comprehensively study interaction networks and their assemblies. However, the study of these interactomes has been hampered by severe methodological limitations. In particular, the affinity purification of intact compl...
Source: Springer protocols feed by Microbiology - October 21, 2015 Category: Microbiology Source Type: news
A Versatile Procedure to Generate Genome-Wide Spatiotemporal Program of Replication in Yeast Species
Here, we describe a complete protocol, comprising both the experimental and the analytical procedures, that allows to generate genome-wide spatiotemporal program of replication and to find the location of chromosomally active replication origins in yeast. The first step consists on synchronizing a cell population by physical discrimination of G1 cells according to their sedimentation coefficient. G1 cells are then synchronously released into S-phase and time-point samples are regularly taken until they reach the G2 phase. Progression through the cell cycle is monitored by measuring DNA content variation by flow cytometry. ...
Source: Springer protocols feed by Microbiology - October 21, 2015 Category: Microbiology Source Type: news
