Tumor analysis by spatial transcriptomics and single cell RNA-seq
dehydration
DEHYDRATION
Spatial transcriptomics will be performed on FFPE tissue sections using the 10X Genomics Visium Spatial Gene Expression platform (10X Genomics, GenomEast, IGBMC, Illkirch, France). Histological sections will be stained using hematoxylin and eosin (H&E) to select the most relevant areas for analysis. Using a Leica RM2235 microtome (Leica, Nanterre, France), tissue sections will be cut from FFPE tissue blocks and placed within the etched frames of the capture areas on the active surface of the Visium Spatial Slide using the Visium CytAssist. The slide will be dried in an oven overnight at 37 °C and then at 60 °C for 25 min before dehydration in ethanol and H&E staining. The slide will finally be transferred to water and mounted in 87% glycerol before image acquisition on the same day. Image mosaic of each section will be acquired to generate a brightfield image of each section including the fiducial marker frame. Subsequently, stained sections will be processed as described in the demonstrated protocol “Visium Spatial Gene-Expression Reagent kits for FFPE, UserGuide” (CG000407). This kit enables spatial quantification of the expression of approximately 18,000 genes on FFPE tissue sections using a pair of specific probes for each targeted gene. Slides containing 4 capture zones will be used. Each 6.5 × 6.5 mm capture zone contains ~ 5,000 spots, and each spot is covered with capture probes bearing a unique barcode, known as a spatial barcode. Quantification and quality control of the final libraries will be performed using Bioanalyzer 2100 (Agilent Technologies, Santa Clara, United States).Libraries will be sequenced on an Illumina NextSeq 2000 platform as paired-end 28 + 50 bases reads. Sequencing data will be processed using the Space Ranger software (10 × Genomics) for analyzing and visualizing spatial gene data produced by the 10 × Genomics Visium Platform including detection tissue, alignment reads, generation of read counts matrices, clustering and gene expression analysis, and place spots in spatial context on the microscope slide image. Differentially expressed gene (DEG) analysis between responders and non-responders will be performed using pseudo-bulking on DESeq2.Signaling pathways and cellular/molecular processes characterized by the expression of genes identified in each spot will be identified using online bioinformatics tools (such as Database for Annotation, Visualization and Integrated Discovery (DAVID) and Search Tool for Retrieval of Interacting Genes/Proteins (STRING)).Single cell RNA-seq analysis will also be performed on the same fixed tissues. Advances in technology have enabled RNA extraction from FFPE which had been a challenge due to RNA modification and degradation [