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13 ARTICLES PUBLISHED IN JoVE

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Neuroscience

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain
Zhiqiang Dong 1, Mahendra Wagle 1, Su Guo 1
1Department of Bioengineering and Therapeutic Sciences, Programs in Human Genetics and Biological Sciences , University of California San Francisco

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

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Bioengineering

Production and Targeting of Monovalent Quantum Dots
Daeha Seo *1,2,3, Justin Farlow *4,5,6, Kade Southard 1,4,7, Young-wook Jun 1,7, Zev J. Gartner 4,5,6,7
1Department of Otolaryngology, University of California, San Francisco, 2Department of Chemistry, University of California, Berkeley, 3Materials Science Division, Lawrence Berkeley National Laboratory, 4Department of Pharmaceutical Chemistry, University of California, San Francisco, 5Tetrad Graduate Program, University of California, San Francisco, 6Center for Systems and Synthetic Biology, University of California, San Francisco, 7Chemistry and Chemical Biology Graduate Program, University of California, San Francisco

We provide detailed instructions for the preparation of monovalent targeted quantum dots (mQDs) from phosphorothioate DNA of defined length. DNA wrapping occurs in high yield, and therefore, products do not require purification. We demonstrate the use of the SNAP tag to target mQDs to cell-surface receptors for live-cell imaging applications.

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Bioengineering

Double Emulsion Generation Using a Polydimethylsiloxane (PDMS) Co-axial Flow Focus Device
Russell H. Cole 1, Tuan M. Tran 2, Adam R. Abate 3,4
1Department of Pharmaceutical Chemistry, University of California, San Francisco, 2Joint UCSF/UCB Bioengineering Graduate Group, University of California, San Francisco, 3Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, 4California Institute for Quantitative Biosciences, University of California, San Francisco

Microfluidic double emulsions generation typically involves devices with patterned wettability or custom-fabricated glass components. Here we describe the fabrication and testing of an all polydimethylsiloxane (PDMS) double emulsion generator that does not require surface treatment or complicated fabrication processes, and is capable of producing double emulsions down to 14 µm.

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Bioengineering

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers
Russell H. Cole 1, Zev J. Gartner 1, Adam R. Abate 2
1Department of Pharmaceutical Chemistry, University of California, San Francisco, 2Department of Bioengineering and Therapeutic Sciences, California Institute for Quantitative Biosciences, University of California, San Francisco

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

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Genetics

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms
Behnom Farboud *1,2, Erin Jarvis *1, Theodore L. Roth *3,4,5,6, Jiyung Shin *1,3, Jacob E. Corn 1,3, Alexander Marson 3,5,6,7,8,9, Barbara J. Meyer 1,2, Nipam H. Patel 1,10, Megan L. Hochstrasser 3
1Department of Molecular Cell Biology, University of California, Berkeley, 2Howard Hughes Medical Institute, University of California, Berkeley, 3Innovative Genomics Institute, University of California, Berkeley, 4Biomedical Sciences Graduate Program, University of California, San Francisco, 5Department of Microbiology and Immunology, University of California, San Francisco, 6Diabetes Center, University of California, San Francisco, 7Chan Zuckerberg Biohub, 8Department of Medicine, University of California, San Francisco, 9UCSF Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, 10Department of Integrative Biology, University of California, Berkeley

Utilizing a preassembled Cas9 ribonucleoprotein complex (RNP) is a powerful method for precise, efficient genome editing. Here, we highlight its utility across a broad range of cells and organisms, including primary human cells and both classic and emerging model organisms.

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Bioengineering

An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing
Benjamin Demaree 1,2, Daniel Weisgerber 1, Freeman Lan 1,2, Adam R. Abate 1,2,3
1Department of Bioengineering and Therapeutic Sciences, California Institute for Quantitative Biosciences, University of California, San Francisco, 2UC Berkeley-UCSF Graduate Program in Bioengineering, University of California, San Francisco, 3Chan Zuckerberg Biohub

Single-cell sequencing reveals genotypic heterogeneity in biological systems, but current technologies lack the throughput necessary for the deep profiling of community composition and function. Here, we describe a microfluidic workflow for sequencing >50,000 single-cell genomes from diverse cell populations.

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Immunology and Infection

Use of Single Chain MHC Technology to Investigate Co-agonism in Human CD8+ T Cell Activation
Xiang Zhao 1, Maryam Hamidinia 1, Joanna Ai Ling Choo 1, Chien Tei Too 1,2, Zi Zong Ho 3, Ee Chee Ren 4, Antonio Bertoletti 3, Paul A. MacAry 1,2,5, Keith G. Gould 6, Joanna Brzostek 1, Nicholas R.J. Gascoigne 1,2,5
1Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, 2Immunology Programme, Life Sciences Institute, National University of Singapore, 3Emerging Infectious Diseases Program, Duke-NUS Graduate Medical School, 4Singapore Immunology Network, A*STAR, 5NUS Graduate School for Integrative Sciences and Engineering (NGS), National University of Singapore, 6Department of Immunology, Wright-Fleming Institute, Imperial College London

This protocol describes the use of single chain MHC class I complexes to investigate molecular interactions in human CD8+ T cell activation: generation of engineered antigen presenting cells expressing single chain constructs, culture of human CD8+ T cell clone and T cell activation experiments.

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Bioengineering

High Throughput Yeast Strain Phenotyping with Droplet-Based RNA Sequencing
Jesse Q. Zhang 1,2, Kai-Chun Chang 1, Leqian Liu 1, Zev J. Gartner 3,5, Adam R. Abate 1,4,5
1Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, 2University of California Berkeley-UCSF Graduate Program in Bioengineering, University of California San Francisco, 3Department of Pharmaceutical Chemistry, University of California San Francisco, 4California Institute for Quantitative Biosciences, University of California San Francisco, 5Chan Zuckerberg Biohub

A bottleneck in the ‘design-build-test’ cycle of microbial engineering is the speed at which we can perform functional screens of strains. We describe a high-throughput method for strain screening applied to hundreds to thousands of yeast cells per experiment that utilizes droplet-based RNA sequencing.

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Bioengineering

Simple, Affordable, and Modular Patterning of Cells using DNA
Katelyn A. Cabral 1, David M. Patterson 2, Olivia J. Scheideler 1, Russell Cole 3, Adam R. Abate 4,5,6, David V. Schaffer 7,8, Lydia L. Sohn 9, Zev J. Gartner 2,6,10
1Graduate Program in Bioengineering, University of California San Francisco and University of California Berkeley, 2Department of Pharmaceutical Chemistry, University of California San Francisco, 3Scribe Biosciences, 4Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, 5California Institute for Quantitative Biosciences, University of California San Francisco, 6Chan Zuckerberg Biohub, University of California San Francisco, 7Department of Chemical & Biomolecular Engineering, University of California Berkeley, 8Helen Wills Neuroscience Institute, University of California Berkeley, 9Department of Mechanical Engineering, University of California Berkeley, 10Center for Cellular Construction, University of California San Francisco

Here we present a protocol to micropattern cells at single-cell resolution using DNA-programmed adhesion. This protocol uses a benchtop photolithography platform to create patterns of DNA oligonucleotides on a glass slide and then labels cell membranes with commercially available complementary oligonucleotides. Hybridization of the oligos results in programmed cell adhesion.

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Biology

Particle Templated Emulsification enables Microfluidic-Free Droplet Assays
Daniel W. Weisgerber 1, Makiko N. Hatori 1, Adam R. Abate 1,2
1Department of Bioengineering and Therapeutic Sciences, California Institute for Quantitative Biosciences, University of California, 2Chan Zuckerberg Biohub

Water-in-oil droplet assays are useful for analytical chemistry, enzyme evolution, and single cell analysis, but typically require microfluidics to form the droplets. Here, we describe particle templated emulsification, a microfluidic-free approach to perform droplet assays.

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Biochemistry

"Cell Surface Capture" Workflow for Label-Free Quantification of the Cell Surface Proteome
Akul Naik 1, Sanjeeva Srivastava 1,2, Arun P. Wiita 1,3,4
1Department of Laboratory Medicine, University of California, San Francisco, 2Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, 3Deptartment of Bioengineering and Therapeutic Sciences, University of California, San Francisco, 4Chan Zuckerberg Biohub

Here, we describe a proteomics workflow for characterization of the cell surface proteome of various cell types. This workflow includes cell surface protein enrichment, subsequent sample preparation, analysis using an LC-MS/MS platform, and data processing with specialized software.

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Methods For Visualizing Intracellular Organelles
Xiang Zhao 1,2, Su Guo 1,2,3
1Chan Zuckerberg Biohub, 2Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, 3Programs in Human Genetics and Biological Sciences, University of California, San Francisco

Methods For Visualizing Intracellular Organelles

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Developmental Biology

Antibody Uptake Assay for Tracking Notch/Delta Endocytosis During the Asymmetric Division of Zebrafish Radial Glia Progenitors
Xiang Zhao 1,2, Su Guo 2,3
1Chan Zuckerberg Biohub, 2Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, 3Programs in Human Genetics and Biological Sciences, University of California San Francisco

This work develops an antibody uptake assay for imaging intra-lineage Notch/DeltaD signaling in dividing radial glia progenitors of the embryonic zebrafish brain.

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