The single-cell RNA sequencing for a-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 a-gustducin. Recent studies revealed that the taste response profiles of a-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 a-gustducin-expressing taste cells in both fungiform and circumvallatae papillae with transgenic mice expressing green fluorescent protein (GFP). Overall design: Transcriptome profile of 90 a-gustducin-positive taste cells of a-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 < of genes, > 0.6% of mitochondrial genes, > 0.1% of Ribosome RNA, and 0.9 < of log3 value of Gnat3 ,that encodes a-gustducin, expression level were further processed.