The Francis Crick Institute 7 articles published in JoVE Biology Imaging ATG9A, a Multi-Spanning Membrane Protein Alexander R. van Vliet*1,2, Stefano De Tito*1, Eugenia Almacellas*1, Sharon A. Tooze1 1Molecular Cell Biology of Autophagy, The Francis Crick Institute, 2MRC Laboratory of Molecular Biology This protocol describes various methods that can help in the study of ATG9A biology, including immunofluorescence followed by image analysis, transient overexpression considerations, and investigating the ATG9A glycosylation status using western blot. Neuroscience Fluorescence-Activated Nuclei Negative Sorting of Neurons Combined with Single Nuclei RNA Sequencing to Study the Hippocampal Neurogenic Niche Thomas Kerloch1, Tjaša Lepko2, Kirill Shkura2, François Guillemot1, Sébastien Gillotin2 1The Francis Crick Institute, 2MSD R&D Innovation Centre Presented here is a method to sequence single nuclei isolated from the mouse dentate gyrus that excludes most neurons through fluorescence-activated nuclei (FAN)-sorting. This approach generates high-quality expression profiles and facilitates the study of most other cell types represented in the niche, including scarce populations such as neural stem cells. Immunology and Infection Analysis of SAMHD1 Restriction by Flow Cytometry in Human Myeloid U937 Cells Paula Ordonez1, Kate N. Bishop2, Jonathan P. Stoye1, Harriet Cordelia Theed Groom3 1Retrovirus-Host Interactions Laboratory, The Francis Crick Institute, 2Retroviral Replication Laboratory, The Francis Crick Institute, 3Sidney Sussex College, Department of Medicine, University of Cambridge Described here is an established method to determine the extent of HIV-1 restriction by the cellular inhibitory protein SAMHD1. Human myeloid lineage U937 cells are transduced with a SAMHD1 expression vector co-expressing YFP, differentiated and then challenged with HIV-RFP. The level of restriction is determined by flow cytometry analysis. Developmental Biology Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages Federica Mangione1,2, Enrique Martin-Blanco1 1Instituto de Biología Molecular de Barcelona, Consejo Superior de Investigaciones Científicas, Parc Científic de Barcelona, 2The Francis Crick Institute This protocol is designed for the imaging and analysis of the dynamics of cell orientation and tissue growth in the Drosophila abdominal epithelia as the fruit fly undergoes metamorphosis. The methodology described here can be applied to the study of different developmental stages, tissues, and subcellular structures in Drosophila or other model organisms. Neuroscience Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains Myrto Denaxa1,2, Guilherme Neves3, Juan Burrone3, Vassilis Pachnis1 1Nervous System Development and Homeostasis Laboratory, The Francis Crick Institute, 2Neuroscience Centre, Biomedical Sciences Research Centre "Al. Fleming", 3 Here we present a protocol, designed to use chemogenetic tools to manipulate the activity of cortical interneuron progenitors transplanted into the cortex of early postnatal mice. Immunology and Infection Optimization of a Quantitative Micro-neutralization Assay Yipu Lin1, Yan Gu1, John W. McCauley1 1Mill Hill Laboratory, The Francis Crick Institute This study describes an imaging-based micro-neutralization assay to analyze the antigenic relationships between viruses. The protocol employs a flatbed scanner and has four steps, including titration, titration quantitation, neutralization, and neutralization quantitation. The assay works well with current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses. Biology Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites Erik Serrao1, Peter Cherepanov2, Alan N. Engelman1 1Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, 2Chromatin Structure and Mobile DNA, The Francis Crick Institute We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.
