PT  - JOURNAL ARTICLE
AU  - James A. Gregory
AU  - Emily Hoelzli
AU  - Rawaan Abdelaal
AU  - Catherine Braine
AU  - Miguel Cuevas
AU  - Madeline Halpern
AU  - Natalie Barretto
AU  - Nadine Schrode
AU  - Güney Akbalik
AU  - Kristy Kang
AU  - Esther Cheng
AU  - Kathryn Bowles
AU  - Steven Lotz
AU  - Susan Goderie
AU  - Celeste M. Karch
AU  - Sally Temple
AU  - Alison Goate
AU  - Kristen Brennand
AU  - Hemali Phatnani
TI  - Cell-type specific gene expression profiling in heterogeneous <em>in vitro</em> cultures using epitope-tagged RPL22
AID  - 10.1101/2020.04.30.064709
DP  - 2020 Jan 01
TA  - bioRxiv
PG  - 2020.04.30.064709
4099  - http://biorxiv.org/content/early/2020/05/01/2020.04.30.064709.short
4100  - http://biorxiv.org/content/early/2020/05/01/2020.04.30.064709.full
AB  - Human induced pluripotent stem cells (hiPSCs) allow the production of defined, disease relevant cell types for transcriptomic and functional studies. A major challenge for hiPSCs disease modeling is to more closely mimic heterocellular networks observed in vivo. As hiPSC cultures become more complex, measuring cell-type specific transcriptomics will become more challenging. Here we report an extension of the RiboTag system, which uses cell-type restricted expression of epitope-tagged RPL22 in mouse tissue, applied to immortalized cell lines, primary mouse astrocytes, and hiPSC-derived neural cells. We constructed lentiviral vectors expressing epitope-tagged RPL22 using constitutive and inducible promoters fused to HA, Flag, or V5 affinity tags in combination with fluorescent reporters; differential expression of uniquely tagged RPL22 in specific cell types allows mRNA from each population to be biochemically purified from mixed cultures. RiboTag expression enables efficient depletion of off-target RNA in mixed species co-cultures of immortalized cell lines using HA and V5 RiboTags. In monocultures of hiPSC-derived neural progenitor cells, motor neurons, and GABAergic neurons expressing RiboTags, we observed an enrichment of protein coding genes and concomitant decrease in mitochondrial and non-coding genes in immunoprecipitated RNA. Depletion efficiency varied across independent replicates of mixed-species co-cultures of hiPSC-derived motor neurons and primary mouse astrocytes. The challenges and potential of implementing RiboTags in complex in vitro cultures are discussed.Competing Interest StatementThe authors have declared no competing interest.