The single-cell RNA sequencing for α-gustducin-positive taste cells. The single-cell RNA sequencing for α-gustducin-positive taste cells

Taste buds are complex sensory organs that are embedded in the epithelium of fungiform papillae (FP) and circumvallate papillae (CV). The sweet, bitter, and umami taste are sensed by type II taste cells that expressed taste receptors (Tas1rs and Tas2rs) which coupled with taste G-protein α-gustducin. Recent studies revealed that the taste response profiles of α-gustducin-expressing cells are different between FP and CV. We applied the high-throughput single-cell RNA-sequencing combined with fluorescence-activated cell sorting (FACS) to profile the transcriptome of the α-gustducin-expressing taste cells in both fungiform and circumvallatae papillae with transgenic mice expressing green fluorescent protein (GFP). Overall design: Transcriptome profile of 90 α-gustducin-positive taste cells of α-gustducin-GFP mice that were collected by fluorescence-activated cell sorting. Single-cell capture and cDNA synthesis were performed by the BD Rhapsody Single-Cell Analysis System (BD). Total cDNA was amplified by the TAS-Seq method (Immunogenetics). Sequencing was performed using Novaseq6000 (Illumina). Raw reads were aligned to the mouse reference sequencing RNA using the Bowtie2 program. Quality control was performed using the DropletUtils package. The expression data matrix was adjusted using distribution-based error correction, which is manufacturer-developed algorithms correcting for sequencing errors. Downstream analyses were performed using the Seurat v2.3.4 R package. The data matrix contained cells derived from FP or CV were integrated. Cells with 500 0.6% of mitochondrial genes, > 0.1% of Ribosome RNA, and 0.9 < of log3 value of Gnat3 ,that encodes α-gustducin, expression level were further processed.

味蕾是嵌入菌状乳头（fungiform papillae, FP）与轮廓乳头（circumvallate papillae, CV）上皮中的复杂感觉器官。甜、苦、鲜味的感知由II型味觉细胞介导，这类细胞表达味觉受体（Tas1rs与Tas2rs），并与味觉G蛋白α-味导素（α-gustducin）偶联。近期研究发现，表达α-味导素的细胞的味觉响应谱在菌状乳头与轮廓乳头之间存在差异。本研究采用高通量单细胞RNA测序结合荧光激活细胞分选（fluorescence-activated cell sorting, FACS）技术，对表达绿色荧光蛋白（green fluorescent protein, GFP）的转基因小鼠中，菌状乳头及轮廓乳头内表达α-味导素的味觉细胞的转录组进行了分析。整体实验设计：通过荧光激活细胞分选收集α-味导素-GFP转基因小鼠的90个α-味导素阳性味觉细胞，开展转录组分析。单细胞捕获与cDNA合成采用BD Rhapsody单细胞分析系统（BD）完成。总cDNA通过TAS-Seq方法（Immunogenetics）进行扩增。测序使用Illumina Novaseq6000平台完成。原始reads通过Bowtie2程序比对至小鼠参考转录组。质控分析采用DropletUtils软件包完成。表达矩阵通过基于分布的误差校正算法进行调整——该算法为仪器自研算法，用于校正测序误差。后续分析采用Seurat v2.3.4 R软件包完成。整合了源自菌状乳头或轮廓乳头的细胞所构成的数据矩阵。后续进一步筛选的细胞需满足以下条件：线粒体基因占比<0.6%、核糖体RNA占比>0.1%，且编码α-味导素的Gnat3基因的log3转换后表达值>0.9。