Seq-Scope: Submicrometer-resolution spatial barcoding technology that enables microscopic examination of tissue transcriptome at single cell and subcellular levels

Purpose: we propose Sequence-Scope (Seq-Scope), which can generate ultra-high definition images of sequence-based molecular signatures resolved at a submicrometer scale. Experimental Methods: Seq-Scope experiment is divided into two consecutive sequencing steps: 1st-Seq and 2nd-Seq. 1st-Seq of Seq-Scope starts with the solid-phase amplification of a single-stranded synthetic oligonucleotide library using an Illumina sequencing-by-synthesis (SBS) platform. 2nd-Seq of Seq-scope begins with overlaying the tissue section slice onto the HDMI-array. Computational Methods: Tissue boundaries are detected by using a custom python code to draw a smoothed density plot to visualize the density of HiSeq reads in a given XY space of each tile. Digital gene expression (DGE) matrices are generate using STAR/STARsolo 2.7.5c with Gene,GeneFull, Velocyto, and polyAtrimming options. Data binning is performed by dividing the imaging space into 100 m2 square grid with 10 m simple side or 25 m2 square grid with 5 m side and collapsing all HDMI-UMI information into one barcode. Binned DGE matrix was analyzed in the Seurat v4 package for clustering analysis. A total of 2 Seq-Scope experiments (colon and liver) were run. Livers were collected from 8 week-old control (Depdc5F/F/Tsc1F/F, male) and TD (Alb-Cre/Depdc5F/F/Tsc1F/F, female) mice. Colons are from 8-week-old C57BL/6 wild-type male mice.