Figure 1. Clustering of bipolar cells by Drop-seq.

(A) Sketch of retinal cross-section depicting major resident cell classes. Rod and cone photoreceptors detect and transduce light stimuli into chemical signals, relaying this information to rod and cone bipolar cells (BCs), respectively (turquoise and purple/orange). BCs synapse on retinal ganglion cells (whose axons form the optic nerve) in the inner plexiform layer (IPL) at varying depths that depend on the BC type.

(B) Overview of experimental strategy. Retinas from Vsx2-GFP mice were dissociated, followed by FAC sorting for GFP+ cells. Single cell libraries were prepared using Drop-seq and sequenced. Raw reads were processed to obtain a digital expression matrix (genes x cells). PCA, followed by graph clustering was used to partition cells into clusters, and identify cluster-specific markers, which were validated in vivo using methods that detect gene expression and cellular morphology in combination.

(C)–(E) 2D visualization of single cell clusters using tSNE. Individual points correspond to single cells colored according to clusters identified by the (C) Louvain-Jaccard, and (D) Infomap algorithms, and numbered in decreasing order of size. Arrows in panels (C), (D) indicate a Louvain-Jaccard BC cluster that was partitioned by Infomap (examined in Figure 5). Panel (E) shows the clustering output of Infomap when applied on cells from a single Drop-seq experiment (50% of the dataset). The tSNE representation was only used for visualization, and not for defining clusters.

(F) Gene expression patterns (columns) of major retinal class markers (left panels) and known BC type markers (right panels) in BC (upper panels) and non-BC clusters (lower panels) based on the clusters in panel C. Clusters with cell-doublet signatures, and that contained < 50 cells are not shown. Putative cell type assignments, based on the expression of known genes, are indicated on the right (see Table S2). Nomenclature for BC types 1 and 5 is based on results in Figures 3 and 4. The size of each circle depicts the percentage of cells in the cluster in which the marker was detected (≥1 UMI), and its color depicts the average transcript count in expressing cells (nTrans). MG = Müller glia, AC = amacrine cells, PR = photoreceptors).

(G) Hierarchical clustering of average gene signatures of BC clusters based (euclidean distance metric, average linkage). The confidence level of each split was assessed using bootstrap (Methods and Resources). Relatedness between clusters was used in prospective cluster assignment to BC type in panel F.

See also Figures S1–S3 and Tables S1–S2