Wellcome Trust Sanger Institute

8 ARTICLES PUBLISHED IN JoVE

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.

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.

Bioengineering

Flexural Rigidity Measurements of Biopolymers Using Gliding Assays

Douglas S. Martin¹, Lu Yu¹, Brian L. Van Hoozen¹

¹Department of Physics, Lawrence University

A method to measure the persistence length or flexural rigidity of biopolymers is described. The method uses a kinesin-driven microtubule gliding assay to experimentally determine the persistence length of individual microtubules and is adaptable to actin-based gliding assays.

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.

Genetics

A Fast and Quantitative Method for Post-translational Modification and Variant Enabled Mapping of Peptides to Genomes

Christoph N. Schlaffner^1,2,3, Georg J. Pirklbauer², Andreas Bender³, Judith A.J. Steen¹, Jyoti S. Choudhary^2,4

¹Department of Neurobiology, F. M. Kirby Neurobiology Center, Boston Children's Hospital, Harvard Medical School, ²Proteomic Mass Spectrometry, Wellcome Trust Sanger Institute, Wellcome Genome Campus, ³Centre for Molecular Informatics, Department of Chemistry, University of Cambridge, ⁴Functional Proteomics Group, Chester Beatty Laboratories, Institute of Cancer Research

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.

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. Lees^1,2, Jessica L. Forbester^1,3, Sally Forrest², Leanne Kane¹, David Goulding¹, Gordon Dougan^1,2

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

Cell Surface Receptor Identification Using Genome-Scale CRISPR/Cas9 Genetic Screens

Sumana Sharma^1,2, 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. Miska^2,3,4, 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.