title: Spatial transcriptomics of homo sapiens: tertiary lymphoid structures (TLS) in pancreatic ductal adenocarcinoma (PDAC) immunotherapy treated
design: A total of 16 pancreatic cancer samples resected from GVAX-treated PDAC patients (clinical trial #NCT00727441) were embedded in OCT media and freshly frozen. Sections were cut into 6.5 x 6.5 mm dimensions compatible with Visium spatial transcriptomics slides. The frozen cohort contained 14 tumor sections and 2 adjacent lymph nodes. We used 4 Visium slides and covered the 16 capture areas with sections cut in a cryostat at 10 µm. The fresh frozen Visium protocol was followed according to manufacturer's recommendations (10x Genomics Spatial 3' v1). Tissue permeabilization and library construction was carried out according to the manufacturer's protocols. Permeabilization time was optimized to 30 minutes. The captured tissue sections were permeabilized to allow RNA to be captured onto the slide where it reverse-transcribed to cDNA. The cDNA was then amplified, and sequencing libraries were prepared using the 10x Genomics Visium Spatial Gene Expression Reagent Kit for Library Preparation. The libraries were then sequenced using an Illumina NovaSeq sequencer. The raw data obtained from the sequencing were processed using the SpaceRanger (spaceranger-1.2.0) pipeline for alignment onto the GRCh38-2020-A reference transcriptome quantification of gene expression.