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Accession IconSRP165642

Biology and Bias in Cell Type-Specific RNAseq of Nucleus Accumbens Medium Spiny Neurons

Organism Icon Mus musculus
Sample Icon 43 Downloadable Samples
Technology Badge IconIllumina HiSeq 4000

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Description
Isolation of cell populations is untangling complex biological interactions, but studies comparing methodologies lack in vivo complexity and draw limited conclusions about the types of transcripts identified by each technique. Furthermore, few studies compare FACS-based techniques to ribosomal affinity purification, and none do so genome-wide. We addressed this gap by systematically comparing nuclear-FACS, whole cell-FACS, and RiboTag affinity purification in the context of D1 or D2 dopamine receptor-expressing medium spiny neuron (MSN) subtypes of the nucleus accumbens (NAc), a key brain reward region. We find that nuclear-FACS-seq generates a substantially longer list of differentially expressed genes between these cell types, and a significantly larger number of neuropsychiatric GWAS hits than the other two methods. RiboTag-seq has much lower coverage of the transcriptome than the other methods, but very efficiently distinguishes D1- and D2-MSNs. We also demonstrate differences between D1- and D2-MSNs with respect to RNA localization, suggesting fundamental cell type differences in mechanisms of transcriptional regulation and subcellular transport of RNAs. Together, these findings guide the field in selecting the RNAseq method that best suits the scientific questions under investigation. Overall design: Forty-nine samples constituting 39 samples from male mice: 16 whole cell-FACS (D1 n=9, D2 n=7), 11 nuclear-FACS (D1 n=6, D2 n=5), and 12 RiboTag (D1 n=6, D2 n=6), and 10 samples from female mice (D1 n=5, D2 n=5).
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49
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