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      Isolation of Highly Purified and Viable Retinal Endothelial Cells

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          Abstract

          The neonatal mouse retinal vascularization model has been widely used in the vascular biology field to investigate mechanisms of angiogenesis and arterial-venous fate specification during blood vessel formation and maturation. Recent advances in next-generation sequencing can further elucidate mechanisms of blood vessel formation and remodeling in this, as well as other, vascular development models. However, an optimized method for isolating retinal endothelial cells that limits tissue digestion-induced cell damage is required for next-generation sequencing applications. In this study, we established a method for isolating neonatal retinal endothelial cells that optimizes cell viability and purity. The CD31+/CD45− endothelial cell population was fluorescence-activated cell sorting (FACS)-isolated from digested postnatal retinas, found to be highly enriched for endothelial cell gene expression, and exhibited no change in viability for 60 min post-FACS. Thus, this method for retinal endothelial cell isolation is compatible with next-generation sequencing applications. Combining this isolation method with next-generation sequencing will enable further delineation of mechanisms underlying vascular development and maturation.

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          Most cited references 49

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          Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets.

          Cells, the basic units of biological structure and function, vary broadly in type and state. Single-cell genomics can characterize cell identity and function, but limitations of ease and scale have prevented its broad application. Here we describe Drop-seq, a strategy for quickly profiling thousands of individual cells by separating them into nanoliter-sized aqueous droplets, associating a different barcode with each cell's RNAs, and sequencing them all together. Drop-seq analyzes mRNA transcripts from thousands of individual cells simultaneously while remembering transcripts' cell of origin. We analyzed transcriptomes from 44,808 mouse retinal cells and identified 39 transcriptionally distinct cell populations, creating a molecular atlas of gene expression for known retinal cell classes and novel candidate cell subtypes. Drop-seq will accelerate biological discovery by enabling routine transcriptional profiling at single-cell resolution. VIDEO ABSTRACT.
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            Single-cell sequencing reveals dissociation-induced gene expression in tissue subpopulations

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              Single-cell chromatin accessibility reveals principles of regulatory variation

              Cell-to-cell variation is a universal feature of life that impacts a wide range of biological phenomena, from developmental plasticity 1,2 to tumor heterogeneity 3 . While recent advances have improved our ability to document cellular phenotypic variation 4–8 the fundamental mechanisms that generate variability from identical DNA sequences remain elusive. Here we reveal the landscape and principles of cellular DNA regulatory variation by developing a robust method for mapping the accessible genome of individual cells via assay for transposase-accessible chromatin using sequencing (ATAC-seq). Single-cell ATAC-seq (scATAC-seq) maps from hundreds of single-cells in aggregate closely resemble accessibility profiles from tens of millions of cells and provides insights into cell-to-cell variation. Accessibility variance is systematically associated with specific trans-factors and cis-elements, and we discover combinations of trans-factors associated with either induction or suppression of cell-to-cell variability. We further identify sets of trans-factors associated with cell-type specific accessibility variance across 8 cell types. Targeted perturbations of cell cycle or transcription factor signaling evoke stimulus-specific changes in this observed variability. The pattern of accessibility variation in cis across the genome recapitulates chromosome topological domains 9 de novo, linking single-cell accessibility variation to three-dimensional genome organization. All together, single-cell analysis of DNA accessibility provides new insight into cellular variation of the “regulome.”
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                Author and article information

                Journal
                JVR
                J Vasc Res
                10.1159/issn.1018-1172
                Journal of Vascular Research
                S. Karger AG
                1018-1172
                1423-0135
                2021
                January 2021
                06 October 2020
                : 58
                : 1
                : 49-57
                Affiliations
                aCardiovascular Research Center, University of Virginia School of Medicine, Charlottesville, Virginia, USA
                bDepartment of Cell Biology, University of Virginia School of Medicine, Charlottesville, Virginia, USA
                cDepartment of Cardiology, University of Virginia School of Medicine, Charlottesville, Virginia, USA
                dCardiovascular Research Center, Yale University School of Medicine, New Haven, Connecticut, USA
                Author notes
                *Nicholas W. Chavkin, Cardiovascular Research Center, University of Virginia School of Medicine, 415 Lane Road, Suite 1010, Charlottesville VA 22908 (USA), nwc6qu@virginia.edu
                Article
                510533 J Vasc Res 2021;58:49–57
                10.1159/000510533
                33022674
                © 2020 S. Karger AG, Basel

                Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

                Page count
                Figures: 5, Pages: 9
                Categories
                Methods in Vascular Biology

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