Broad Institute View Institution's Website 8 articles published in JoVE Biology A Single Cell Dissociation Approach for Molecular Analysis of Urinary Bladder in the Mouse Following Spinal Cord Injury Hussein Atta*1,2, Ali Hashemi Gheinani*1,2, Amanda Wacker1, Yaser Heshmati3,4,5, Alex Bigger-Allen1,6, George Lambrinos1,2, Yao Gao2,7, Diane R. Bielenberg2,7, Rosalyn M. Adam1,2 1 The goal of this protocol is to apply an optimized tissue dissociation protocol to a mouse model of spinal cord injury and validate the approach for single cell analysis by flow cytometry. Genetics CRISPR Guide RNA Cloning for Mammalian Systems Sathiji Nageshwaran*1,2, Alejandro Chavez*1,2,3, Nan Cher Yeo1,2, Xiaoge Guo1,2, Alissa Lance-Byrne1, Angela Tung1, James J. Collins1,4,5,6,7, George M. Church1,2 1Wyss Institute for Biologically Inspired Engineering, Harvard University, 2Department of Genetics, Harvard Medical School, 3Department of Pathology, Massachusetts General Hospital, 4Institute for Medical Engineering & Science, Massachusetts Institute of Technology, 5Synthetic Biology Center, Massachusetts Institute of Technology, 6Department of Biological Engineering, Massachusetts Institute of Technology, 7Broad Institute Here, a simple, efficient, and cost-effective method of sgRNA cloning is outlined. Cancer Research Using CRISPR/Cas9 Gene Editing to Investigate the Oncogenic Activity of Mutant Calreticulin in Cytokine Dependent Hematopoietic Cells Nouran S. Abdelfattah1, Ann Mullally1,2,3 1 Targeted gene editing using CRISPR/Cas9 has greatly facilitated the understanding of the biological functions of genes. Here, we utilize the CRISPR/Cas9 methodology to model calreticulin mutations in cytokine-dependent hematopoietic cells in order to study their oncogenic activity. Biochemistry A Simple, Robust, and High Throughput Single Molecule Flow Stretching Assay Implementation for Studying Transport of Molecules Along DNA Kan Xiong1,2, Paul C. Blainey1,2 1Broad Institute, Massachusetts Institute of Technology and Harvard Medical School, 2Dept. of Biological Engineering, Massachusetts Institute of Technology This protocol demonstrates a simple, robust and high throughput single molecule flow-stretching assay for studying one-dimensional (1D) diffusion of molecules along DNA. Developmental Biology Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae Elliott J. Hagedorn1,2, Jennifer L. Cillis3, Caitlyn R. Curley3, Taylor C. Patch3, Brian Li1,2, Bradley W. Blaser1,2,7, Raquel Riquelme1,2, Leonard I. Zon1,2,4,5,6, Dhvanit I. Shah1,2,3,4,5 1Division of Hematology, Department of Medicine, Brigham and Women’s Hospital, 4Harvard Stem Cell Institute, 5Broad Institute of Massachusetts Institute of Technology, 6Howard Hughes Medical Institute, 7Division of Hematologic Malignancies, Dana-Farber Cancer Institute This protocol provides step-by-step instruction on how to generate parabiotic zebrafish embryos of different genetic backgrounds. When combined with the unparalleled imaging capabilities of the zebrafish embryo, this method provides a uniquely powerful means to investigate cell-autonomous versus non-cell-autonomous functions for candidate genes of interest. Biology Simple Bulk Readout of Digital Nucleic Acid Quantification Assays Leanna S. Morinishi1, Paul Blainey2 1Broad Institute, 2Department of Biological Engineering, Massachusetts Institute of Technology We describe an endpoint digital assay for quantifying nucleic acids with a simplified (analog) readout. We measure bulk fluorescence of droplet-based digital assays using a standard qPCR machine rather than specialized instrumentation and confirm our results by microscopy. Biology Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis Alexandra Naba1, Karl R. Clauser2, Richard O. Hynes1 1Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 2Proteomics Platform, Broad Institute This protocol describes a procedure for enriching ECM proteins from tissues or tumors and deglycosylating and digesting the ECM-enriched preparations into peptides to analyze their protein composition by mass spectrometry. Biology Massively Parallel Reporter Assays in Cultured Mammalian Cells Alexandre Melnikov1, Xiaolan Zhang1, Peter Rogov1, Li Wang1, Tarjei S. Mikkelsen1 1Broad Institute The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. Coupling candidate regulatory elements to reporter genes that carry identifying sequence tags enables massive parallelization of these assays.