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

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).

细胞群体分离是厘清复杂生物相互作用的重要手段，但现有针对不同分离方法的比较研究往往未能还原体内环境的复杂性，且对各技术所鉴定的转录本类型所能得出的结论较为有限。此外，鲜有研究对比基于荧光激活细胞分选（Fluorescence-Activated Cell Sorting，FACS）的技术与核糖体亲和纯化技术，且尚无全基因组层面的相关对比研究。为此，我们填补了这一研究空白，针对伏隔核（nucleus accumbens，NAc）——大脑奖赏通路的关键脑区——中表达D1或D2多巴胺受体的中型多棘神经元（medium spiny neuron，MSN）亚型，系统比较了细胞核-FACS、全细胞-FACS以及RiboTag亲和纯化三种技术。我们发现，细胞核-FACS-seq在两类神经元亚型间鉴定出的差异表达基因数量远多于另外两种方法，且所富集的神经精神疾病全基因组关联研究（Genome-Wide Association Study，GWAS）命中基因的数量也显著更多。RiboTag-seq的转录组覆盖度远低于另外两种方法，但能极为高效地区分D1-与D2-MSN。我们还证实了D1与D2-MSN在RNA定位方面存在差异，这提示两类细胞在转录调控机制与RNA亚细胞运输过程中存在根本性的细胞类型差异。综上，本研究结果可为该领域选择最契合研究问题的RNAseq方法提供参考。实验设计：本研究共纳入49个样本，其中39个样本来自雄性小鼠：16个全细胞-FACS样本（D1型n=9，D2型n=7）、11个细胞核-FACS样本（D1型n=6，D2型n=5）以及12个RiboTag样本（D1型n=6，D2型n=6）；另有10个样本来自雌性小鼠（D1型n=5，D2型n=5）。