GSM5708496: PR 6; Homo sapiens; RNA-Seq
For spatial transcriptomics, we used the Visium Spatial Gene Expression Reagent Kit (10X Genomics). Tissue optimization was done using the Visium Spatial Tissue Optimization Slide & Reagent kit. The optimization, as well as the analysis, was performed using tissue at 10-µm thickness per the manufacturer's protocol. The selected permeabilization time was 20 minutes. Bright-field images of the tissue were captured according to the requirements of the manufacturer, using a Nikon Eclipse Ni microscope and Nikon DS-Ri1 8-bit camera. The objective was a Nikon Plan Apo with 10× magnification and 0.45 numerical aperture. We used Nikon Elements software with a resolution of 0.91 µm/pixel. Camera settings were as follows: format 1280×1024 (fine), exposure 10.0 ms (±0.0 EV), analog gain 1.00. The Visium Spatial Gene Expression Slide & Reagent kit was used for library construction. One section per patient (10 µm) was mounted into each capture area. Each capture area on a Visium for FFPE slide has an array containing capture probes that bind to RNA. The probe pairs are extended to incorporate complements of the spatial barcodes, and sequencing libraries are prepared. The libraries are then sequenced and data visualized to determine which genes are expressed and where, as well as in what quantity. A capture area is defined as a 6.5 × 6.5 mm area that contains 5,000 barcoded spots that are 55 µm in diameter and 100 µm center to center between spots, providing an average resolution of 1 to 10 cells (10X Genomics). All following steps (cDNA synthesis and amplification, library construction, and post-library construction quality control) were done according to the manufacturer's protocol. The libraries were sequenced on a NovaSeq 6000 (Illumina).