Wellcome Trust Sanger Institute View Institution's Website 8 articles published in JoVE Genetics Cell Surface Receptor Identification Using Genome-Scale CRISPR/Cas9 Genetic Screens Sumana Sharma1,2, Gavin J. Wright1 1Cell Surface Signalling Laboratory, Wellcome Trust Sanger Institute, 2EMBL-EBI, Wellcome Genome Campus This manuscript describes a genome-scale cell-based screening approach to identify extracellular receptor-ligand interactions. Genetics High-Throughput Quantitative RT-PCR in Single and Bulk C. elegans Samples Using Nanofluidic Technology Laetitia Chauve*1, Jérémie Le Pen*2,3,5, Francesca Hodge1, Pia Todtenhaupt1, Laura Biggins1, Eric A. Miska2,3,4, Simon Andrews1, Olivia Casanueva1 1Babraham Institute, 2Gurdon Institute, University of Cambridge, 3Department of Genetics, University of Cambridge, 4Wellcome Trust Genome Campus, Wellcome Trust Sanger Institute, 5Laboratory of Virology and Infectious Disease, The Rockefeller University In this article a high-throughput protocol for fast and reliable determination of gene expression levels in single or bulk C. elegans samples is described. This protocol does not require RNA isolation and produces cDNA directly from samples. It can be used together with high-throughput multiplexed nanofluidic real-time qPCR platforms. Immunology and Infection Using Human Induced Pluripotent Stem Cell-derived Intestinal Organoids to Study and Modify Epithelial Cell Protection Against Salmonella and Other Pathogens Emily A. Lees1,2, Jessica L. Forbester1,3, Sally Forrest2, Leanne Kane1, David Goulding1, Gordon Dougan1,2 1Wellcome Trust Sanger Institute, 2Department of Medicine, University of Cambridge, 3University of Cardiff Human induced pluripotent stem cell (hiPSC)-derived intestinal organoids offer exciting opportunities to model enteric diseases in vitro. We demonstrate the differentiation of hiPSCs into intestinal organoids (iHOs), the stimulation of these iHOs with cytokines, and the microinjection of Salmonella Typhimurium into the iHO lumen, enabling the study of an epithelial invasion by this pathogen. Genetics A Fast and Quantitative Method for Post-translational Modification and Variant Enabled Mapping of Peptides to Genomes Christoph N. Schlaffner1,2,3, Georg J. Pirklbauer2, Andreas Bender3, Judith A.J. Steen1, Jyoti S. Choudhary2,4 1 Here we present the proteogenomic tool PoGo and protocols for fast, quantitative, post-translational modification and variant enabled mapping of peptides identified through mass spectrometry onto reference genomes. This tool is of use to integrate and visualize proteogenomic and personal proteomic studies interfacing with orthogonal genomics data. Biochemistry Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling Mercedes Pardo1, Daniel Bode1, Lu Yu1, Jyoti S. Choudhary1 1Proteomic Mass Spectrometry, Wellcome Trust Sanger Institute Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes. Immunology and Infection A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting Dumizulu L. Tembo1, Jacqui Montgomery1, Alister G. Craig2, Samuel C. Wassmer3 1Malawi-Liverpool-Wellcome Trust Clinical Research Programme, 2Liverpool School of Tropical Medicine, 3Department of Microbiology, Division of Medical Parasitology, New York University School of Medicine This method investigates the platelet-mediated clumping phenotype of Plasmodium falciparum-infected erythrocytes (pRBC) in clinical isolates. This is performed by isolating and co-incubating platelet-rich plasma and a suspension of pRBC. Biology Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions Jason S. Kerr1, Gavin J. Wright1 1Cell Surface Signalling Laboratory, Wellcome Trust Sanger Institute AVEXIS is a high throughput protein interaction assay developed to systematically screen for novel extracellular receptor-ligand pairs involved in cellular recognition processes. It is specifically designed to detect transient protein interactions that are difficult to identify using other high throughput approaches. Biology iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution Julian Konig1, Kathi Zarnack2, Gregor Rot3, Tomaz Curk3, Melis Kayikci1, Blaz Zupan3, Daniel J. Turner4, Nicholas M. Luscombe2, Jernej Ule1 1Laboratory of Molecular Biology, Medical Research Council - MRC, 2European Bioinformatics Institute, EMBL Heidelberg, 3Computer and Information Science, University of Ljubljana, 4Wellcome Trust Genome Campus, Wellcome Trust Sanger Institute The spatial arrangement of RNA-binding proteins on a transcript is a key determinant of post-transcriptional regulation. Therefore, we developed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) that allows precise genome-wide mapping of the binding sites of an RNA-binding protein.