GSE176392
Single-cell RNA-seq reveals cellular heterogeneity of activated pDCs stimulated by R848
Purpose: PDCs will differentiated into different subclusters upon single stimulation. The goals of this study are to identify the heterogeneity of activated pDCs stimulated by R848. Methods: Human primary plasmacytoid dendritic cells were stimulated with R848 for 16 hours. Then, after FACS sorting, about 12000 pDCs were counted and the viability was confirmed to be >90%. Single-cell suspensions were loaded on a 10x Genomics Chromium single-cell 3' v2 chip to prepare the libraries according to the manufacturer's protocol. The library quality was checked by Agilent 2100 Bioanalyzer and Qubit fluorometric quantitation. Samples were sequenced on an Illumina NovaSeq 6000 with a sequencing depth of at least 50000 reads per cell. The raw fastq data were processed with CellRanger (v2.1.1) by using GRCh38 annotation (v1.2.0) (Zheng, G.X., et al.Nat Commun 2017;8,14049.). Output from CellRanger was loaded into R and used Seurat (v3.1.1) for further analysis (Stuart, T., et al. Cell 2019;177,1888-1902 e1821.). Cells with fewer than 1000 or more than 4500 expressed genes, and a high percentage of mitochondrial genes (>5%) were removed. We also used cell selection as Peter Karchenko-the Pagoda way to define outliers. Among them, 6010 cells passed the filtering. After normalizing and scaling, we used 'Find Variable Features' to identify 1000 highly variable genes. Then we used 992 highly variable genes (removing mitochondrial and ribosomal genes) for PCA. Upon 'Elbow plot', we selected the first 20 PC for clustering and UMAP visualization with default parameters. For clustering, we selected resolution parameter=0.2 which produced 4 clusters. To obtain cluster-specific gene signatures, we identified differential expression analysis of each cluster with default parameters. IFN scores based on IFN gene sets were calculated for each cell as the average of scaled expression of these genes and corrected by subtracting the average of a large set of similar genes, as proposed by Tirosh et al (Tirosh, I., et al. Science 2016;352,189-196.). Results: 4 subcluster of pDCs were obtained.Group 0 express chemokines, group 1 express Gzmb, group 2 express type I interferons, and group 3 express Id2 and IL-23A. Overall design: Single cell analysis of pDC subclusters by using 10×Genomics technology.