Cell Type-Specific In Vitro Gene Expression Profiling of Stem Cell-Derived Neural Models.

Cells
James A GregoryHemali Phatnani

Abstract

Genetic and genomic studies of brain disease increasingly demonstrate disease-associated interactions between the cell types of the brain. Increasingly complex and more physiologically relevant human-induced pluripotent stem cell (hiPSC)-based models better explore the molecular mechanisms underlying disease but also challenge our ability to resolve cell type-specific perturbations. Here, we report an extension of the RiboTag system, first developed to achieve cell type-restricted expression of epitope-tagged ribosomal protein (RPL22) in mouse tissue, to a variety of in vitro applications, including immortalized cell lines, primary mouse astrocytes, and hiPSC-derived neurons. RiboTag expression enables depletion of up to 87 percent of off-target RNA in mixed species co-cultures. Nonetheless, depletion efficiency varies across independent experimental replicates, particularly for hiPSC-derived motor neurons. The challenges and potential of implementing RiboTags in complex in vitro cultures are discussed.

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Methods Mentioned

BETA
fluorescence-activated
laser capture microdissection
scRNA-seq
affinity purification
transfection
FACS
immunoprecipitation
electrophoresis
RNA-seq
flow cytometry

Software Mentioned

samtools
RiboTags
GSEA
Ensembl
fgsea
STAR
Subread package
TrimGalore

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