GSE169706
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 µm2 square grid with 10 µm simple side or 25 µm2 square grid with 5 µm side and collapsing all HDMI-UMI information into one barcode. Binned DGE matrix was analyzed in the Seurat v4 package for clustering analysis. Overall design: 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.