(a) Schematic of Probe-Seq for the adult mouse retina. The retina was dissociated into single cells, fixed, and permeabilized. Cells were incubated with gene-specific probe sets against Vsx2 and Grik1 and subsequently incubated with fluorescent oligos. Three populations of labeled cells (Vsx2-, Vsx2+/Grik1-, and Vsx2+/Grik1+) were isolated by FACS for downstream RNA sequencing. (b) SABER-FISH signals from an adult mouse retina section using Vsx2 and Grik1 probe sets. (c) Representative FACS plot of all events (top panel) on a Hoechst histogram. The debris is the peak near 0. The first peak after the debris is the single cell 2N peak. 4N doublets and other cell clumps are in the peaks to the right of the single cell peak. Representative FACS plots of all single cells (middle panel) with Vsx2 fluorescence on the y-axis and autofluorescence (561 nm) on the x-axis. The negative population ran along the diagonal. The Vsx2+ population (12.1%) was left shifted, indicating high Vsx2 fluorescence and low autofluorescence. FACS plot of only the Vsx2+ population (bottom panel) with Grik1 fluorescence on the y-axis and autofluorescence (561 nm) on the x-axis. Vsx2+/Grik1+ population (16.7%) displayed strong separation from the Vsx2+/Grik1- population (81.2%). The Grik1MID population was included in the Vsx2+/Grik1+ population. (d) Expected distribution of retinal cell type markers expressed in each isolated population. (e) Images of dissociated mouse retinal cells after the SABER-FISH protocol on dissociated mouse retinal cells before FACS. (f) A heatmap representing relative expression levels of BC subtype markers previously identified by scRNA sequencing that are differentially expressed (adjusted p-value<0.05) between Grik1- and Grik1+ populations. (g) A representative scatter plot of log2 normalized counts of all genes (left panel) or BC2 – BC4 marker genes (right panel) between Live Grik1+ cells and Probe-Seq Grik1+ Cells. Red line indicates a slope of 1. Select cell class-specific markers (Opn1mw (cones), Apoe (MG), and Rho (rods)) are labeled in red. (h) A heatmap of the stain index with varying levels of transcript expression and number of oligos. The boundary indicates a cutoff of SI < 2. (i) Schematic and flow cytometry plots of iterative Probe-Seq. Three gene-specific probe sets (Gad1, Vsx2, and Grm6) were hybridized to dissociated mouse retinal cells, and fluorescent oligos were hybridized only to Gad1 and Vsx2 probe sets to detect subsets of ACs (Gad1+; 3.54%) and BC/MG (Vsx2+; 11.9%). The fluorescent oligos were subsequently stripped with 50% formamide, which abolished the staining based on flow cytometry (middle panel). Fluorescent oligos for Grm6 were then hybridized to label a subset of BCs (right panel; Grm6+; 6.78%). HC, Horizontal Cell; RGC, Retinal Ganglion Cell; AC, Amacrine Cell; BC, Bipolar Cell; MG, Müller Glia; ONL, Outer Nuclear Layer; INL, Inner Nuclear Layer; GCL, Ganglion Cell Layer. Scale bars: 10 µm (b, e).