Fig. 4: Application of Light-Seq for spatial barcoding of three main retinal layers in fixed frozen mouse retina sections.
a, Three regions of the mouse retina were uniquely barcoded: the ONL with Barcode 1, the BCL with Barcode 2 and the GCL with Barcode 3. b, After barcoding, fluorescently labeled barcode strands were detected in the targeted cell layers: ONL (magenta, Barcode 1; 1,112 ± 199 cells, n = 4 sections), BCL (cyan, Barcode 2; 298 ± 29 cells, n = 4 sections) and GCL (green, Barcode 3; 91 ± 14 cells, n = 4 sections). c, Volcano plots of differentially expressed genes between the ONL and BCL (top), ONL and GCL (middle) and BCL and GCL (bottom), with select markers labeled. The x and y axes show the log2(fold change) and the log10(P value), respectively. d, Heatmap of z-scores for differentially expressed genes with enrichment in just one layer (Padj < 0.05; see source data; two-sided Wald test with Benjamini–Hochberg adjustment for multiple hypothesis testing). e, Boxplot of estimated sensitivity of Light-Seq (n = 4 replicates, 16 genes) and Drop-Seq46 (n = 6 replicates, 16 genes) compared with smFISH data for bipolar subtype marker genes with measured abundances based on quantitative smFISH50. Sensitivity is defined as (number of expected transcripts by smFISH)/(number of observed reads by Light-Seq or Drop-Seq). Midline marks the median and edges indicate the 25th and 75th percentiles. Whiskers extend to encompass all data not considered outliers (default threshold in MATLAB boxplot function; maximum whisker length is 1.5 × interquartile range). Dot color corresponds to replicate number. f, DAPI, WGA staining and IF for VSX2 and PAX6 proteins were performed on the same tissue section after extraction of barcoded cDNAs. Scale bars are 100 μm in a, and 50 µm (left) and 200 µm (right) in b and f.
