T.chinensis_Integrated
Description
Integrated atlas of T. chinensis retina scRNA-seq. Integrated object (TreeShrew_all_cells_integrated.RDS) was generated using the following GSEs (GSE237213 & GSE227524).
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Steps to reproduce
T.chinensis retinal atlas Preprocessing T. chinensis single cell gene by cell matrices (GSE237213 & GSE227524) were downloaded and processed using either a Sketch-based or PCA-integration Seurat (v5) workflow. Matrices were merged into a single SeuratObject and subsequently normalized using default parameters for normalization (NormalizeData) and variable feature selection (FindVariableFeatures; features = 2000). To integrate the two datasets, we used a sketch-based approach (SketchData; ncells = 5000, method = “LeverageScore”), followed by scaling (ScaleData) and principal component analysis (RunPCA) on the sketch-assay. To integrate these layers, we performed integration (IntegrateLayers; k.anchors = 20, dims = 1:30) via reciprocal PCA using GSE237213 as a reference. We then performed neighbor analysis (FindNeighbors; reduction = “integrated.pca”, dims = 1:30), clustering (FindClusters; resolution = 2), and dimensionality reduction (RunUMAP; reduction = “integrated.pca”, dims = 1:30). Then, the integration and sketch-data were projected onto the non-sketched cells (ProjectIntegration; ProjectData) to identify clusters. Cell class Identification To identify cell classes that correspond to T. chinensis atlas clusters, we projected modules of canonical markers used in (1) onto the integrated atlas. Clusters with mean projected-module scores greater than 0 were assigned to a cell class. When encountered, clusters with two annotations were manually inspected for gene expression of cell class markers and manually assigned to a final class based on expression. Canonical markers included for each cell class are as follows: • RGC markers = RBPMS, RBPMS2, SLC17A6, THY1, NEFM • BC markers = VSX1, OTX2, GRM6, TRPM1 • AC markers = TFAP2A, TFAP2B, TFAP2C, GAD1, GAD2-AS-1, SLC6A9, C1QL1, C1QL2 • HC markers = ONECUT1, ONECUT2, LHX1, CALB1 • PR markers = PDE6H, CRX, ARR3, SAG • MG markers = SLC1A3, RLBP1, APOE • Other markers = S100B, GFAP, C1QA, C1QB, CLDN5, IGFBP7
Institutions
- Cincinnati Children's Hospital Medical Center