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Morgridge Institute for Research

3 ARTICLES PUBLISHED IN JoVE

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Biology

Single Read and Paired End mRNA-Seq Illumina Libraries from 10 Nanograms Total RNA
Srikumar Sengupta 1, Jennifer M. Bolin 1, Victor Ruotti 1, Bao Kim Nguyen 1, James A. Thomson 1,2,3, Angela L. Elwell 1, Ron Stewart 1
1Regenerative Biology, Morgridge Institute for Research, 2Department of Cell & Regenerative Biology, University of Wisconsin, 3Department of Molecular, Cellular, & Regenerative Biology, University of California

Here we describe a method for preparation of both single read and paired end Illumina mRNA-Seq sequencing libraries for gene expression analysis based on T7 linear RNA amplification. This protocol requires only 10 nanograms of starting total RNA and generates highly consistent libraries representing whole transcripts.

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Biochemistry

Micropatterning Transmission Electron Microscopy Grids to Direct Cell Positioning within Whole-Cell Cryo-Electron Tomography Workflows
Bryan S. Sibert *1,2,3, Joseph Y. Kim *1,4, Jie E. Yang 1,2,3, Elizabeth R. Wright 1,2,3,5
1Department of Biochemistry, University of Wisconsin, Madison, 2Cryo-Electron Microscopy Research Center, Department of Biochemistry, University of Wisconsin, Madison, 3Midwest Center for Cryo-Electron Tomography, Department of Biochemistry, University of Wisconsin, Madison, 4Department of Chemistry, University of Wisconsin, Madison, 5Morgridge Institute for Research

The goal of this protocol is to direct cell adhesion and growth to targeted areas of grids for cryo-electron microscopy. This is achieved by applying an anti-fouling layer that is ablated in user-specified patterns followed by deposition of extra-cellular matrix proteins in the patterned areas prior to cell seeding.

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Neuroscience

Classification of Neural Stem Cell Activation State In Vitro using Autofluorescence
Christopher S. Morrow 1, Amani A. Gillette 2,3, Melissa C. Skala 2,3, Darcie L. Moore 1
1Department of Neuroscience, University of Wisconsin-Madison, 2Morgridge Institute for Research, 3Department of Biomedical Engineering, University of Wisconsin-Madison

This protocol describes strategies to identify and enrich for cell-state in primary adult mouse neural stem cell cultures by autofluorescence imaging using i) a confocal microscope, ii) a fluorescent activated cell sorter to perform intensity imaging, or iii) a multiphoton microscope to perform fluorescence lifetime imaging.

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