Helmholtz Zentrum, German Research Center for Environmental Health 5 articles published in JoVE Biochemistry Measuring and Interpreting Oxygen Consumption Rates in Whole Fly Head Segments Louisa Jutta Dietz1, Anuroop Venkateswaran Venkatasubramani1, Annika Müller-Eigner2, Martin Hrabe de Angelis3,4,5, Axel Imhof1, Lore Becker3, Shahaf Peleg2,6,7 1Munich Center of Integrated Protein Science and Biomedical Center, Ludwig-Maximilians University of Munich, 2Laboratory for Metabolism and Epigenetics in Aging, Leibniz Institute for Farm Animal Biology (FBN), 3German Mouse Clinic, Helmholtz Zentrum Munich, German Research Center for Environment and Health (GmbH), 4German Center for Diabetes Research (DZD), 5Chair of Experimental Genetics, School of Life Science Weihenstephan, Technische Universität München, 6Laboratory for Metabolism and Epigenetics in Brain Aging, Institute of Neuroregeneration & Neurorehabilitation of Qingdao University, 7Molecular Biology Division, Biomedical Center, Faculty of Medicine, Ludwig-Maximilians University of Munich Measuring alterations in metabolic rates is central to understanding the progression of various diseases and aging. Here, we present a novel technique to measure whole head oxygen consumption that more closely resembles the physiological state and may aid in revealing novel drugs that modify mitochondrial activity. Genetics A Customizable Protocol for String Assembly gRNA Cloning (STAgR) Christopher T. Breunig1,2, Andrea M. Neuner1,2, Jessica Giehrl-Schwab3, Wolfgang Wurst3, Magdalena Götz2,4, Stefan H. Stricker1,2 1MCN Junior Research Group, Munich Center for Neurosciences, Ludwig Maximilian Universitat, BioMedical Center, 2Institute of Stem Cell Research, Helmholtz Zentrum, German Research Center for Environmental Health, 3Institute of Developmental Genetics, Helmholtz Zentrum, German Research Center for Environmental Health, 4Physiological Genomics, Ludwig Maximilian Universitat, BioMedical Center Here, we present string assembly gRNA cloning (STAgR), a method to easily multiplex gRNA vectors for CRISPR/Cas9 approaches. STAgR makes gRNA multiplexing simple, efficient and customizable. Genetics Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation Kathrin Davari*1, Johannes Lichti*1, Caroline C. Friedel2, Elke Glasmacher1,3 1Institute for Diabetes and Obesity (IDO), German Center for Diabetes Research (DZD), Helmholtz Zentrum München, 2Institute for Informatics, Ludwig-Maximilians-Universität München, 3Roche Pharma Research and Early Development, Large Molecule Research, Roche Innovation Center Penzberg This protocol describes the combinatorial use of ChIP-seq, 4sU-seq, total RNA-seq, and ribosome profiling for cell lines and primary cells. It enables tracking changes in transcription-factor binding, de novo transcription, RNA processing, turnover and translation over time, and displaying the overall course of events in activated and/or rapidly changing cells. Genetics A Universal Protocol for Large-scale gRNA Library Production from any DNA Source Anna Köferle1, Stefan H. Stricker1,2 1MCN Junior Research Group, Munich Center for Neurosciences, Ludwig-Maximilian-Universität, BioMedical Center, 2Epigenetic Engineering, Institute of Stem Cell Research, Helmholtz Zentrum, German Research Center for Environmental Health Methods for generating large-scale gRNA libraries should be simple, efficient and cost-effective. We describe a protocol for the production of gRNA libraries based on enzymatic digestion of target DNA. This method, CORALINA (comprehensive gRNA library generation through controlled nuclease activity) presents an alternative to costly custom oligonucleotide synthesis. Biochemistry Combining Wet and Dry Lab Techniques to Guide the Crystallization of Large Coiled-coil Containing Proteins Jenna K. Zalewski1, Simone Heber1,2, Joshua H. Mo1, Keith O'Conor1, Jeffrey D. Hildebrand1, Andrew P. VanDemark1 1Department of Biological Sciences, University of Pittsburgh, 2Institute of Structural Biology, German Research Center for Environmental Health We describe a framework incorporating straightforward biochemical and computational analysis to guide the characterization and crystallization of large coiled-coil domains. This framework can be adapted for globular proteins or extended to incorporate a variety of high-throughput techniques.