High-resolution RNA capture from tissue by Slide-seq. (A) Left: Schematic of array generation. A monolayer of randomly deposited, DNA barcoded beads (termed a "puck") is spatially indexed by SOLiD sequencing. Top Right: A representative puck with called barcodes shown in black. Bottom Right: A composite image of the same puck colored by the base calls for a single base of SOLiD sequencing. (Scale bar 500 μm) (B) Top Row: Schematic of the sample preparation procedure developed for Slide-seq. Total time for library generation is ~3 hrs. Bottom Row: Schematic of a naïve analysis, in which each bead is clustered by its gene expression, visualized in a tSNE two-dimensional embedding, and by locations in space. (C) Spatial positions of Slide-seq beads, colored by clusters defined purely by gene expression relationships amongst beads, across five tissue types (see Fig. S2 for tSNE embeddings and definitions). (D) Characterization of lateral diffusion of signal on the Slide-seq surface. Top Left: Digital image of a Slide-seq puck with bead color intensity scaled by total transcript counts. Top Right: Image of the adjacent tissue section, stained with DAPI (scale bar 500 μm). Boxes represent regions where an intensity profile was taken across CA1. Bottom left: Profile of pixel intensity across CA1 in Slide-seq. Bottom right: Profile across CA1 in DAPI stained tissue. Red dots represent locations of half max of the distribution. (E) Quantification of full width at half maximum of profiles in (D), from both Slide-seq (red dots) and DAPI-stained tissue (blue dots) (dotted line, mean; N = 10 profiles) (F) Log ratio of total number of quantified RNA transcripts on a puck to the number of cells counted on a serially stained DAPI slice of equal area (dotted line, mean) across five different tissues.