Morgridge Institute for Research

3 ARTICLES PUBLISHED IN JoVE

Biology

Single Read and Paired End mRNA-Seq Illumina Libraries from 10 Nanograms Total RNA

Srikumar Sengupta¹, Jennifer M. Bolin¹, Victor Ruotti¹, Bao Kim Nguyen¹, James A. Thomson^1,2,3, Angela L. Elwell¹, Ron Stewart¹

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.

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.

Neuroscience

Classification of Neural Stem Cell Activation State In Vitro using Autofluorescence

Christopher S. Morrow¹, Amani A. Gillette^2,3, Melissa C. Skala^2,3, Darcie L. Moore¹

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.