Cx3050s cryostat

10x Genomics Visium protocol was applied on optimal cutting temperature medium (OCT)-embedded fresh frozen samples. All tissues were sectioned using the Leica CX3050S cryostat and were cut at 10 µm. The samples were selected on the basis of morphology, orientation (based on H&E) and RNA integrity number that was obtained using Agilent2100 Bioanalyzer. Tissue optimization was performed to obtain permeabilization time for fetal tissue (12 min) and after optimization the Visium spatial gene expression protocol from 10X Genomics was performed using Library Preparation slide and following the manufacturer’s protocol. After transcript capture, Visium Library Preparation Protocol from 10x Genomics was performed. All images for this process were scanned at 40× on Hamamatsu NanoZoomer S60. Eight cDNA libraries were diluted and pooled to a final concentration of 2.25 nM (200 µl volume) and sequenced on 2× SP flow cells of Illumina NovaSeq 6000.

Fresh frozen samples from tumor core and tumour-normal interface tissues were first embedded in optimal cutting temperature medium (OCT) compound and then sectioned into 10 μm-thick sections using the Leica CX3050S cryostat. The generated sections were selected based on H&E staining with focusing on morphology and orientation. A further selection on samples was conducted based on the RNA integrity number obtained from Agilent2100 Bioanalyzer. Tissue optimization was performed respectively on tumor core and tumour-normal interface samples. After optimization, the Visium spatial gene expression protocol from 10X Genomics was performed using the Library Preparation slide and following the manufacturer’s protocol. After transcript capture, Visium Library Preparation was further performed following the manufacturer’s protocol. All images for this process were scanned at 40× on Hamamatsu NanoZoomer S60. cDNA libraries from five tumor core and 11 tumor normal interface samples were diluted and pooled to a final concentration of 2.25 nM (200 μL volume) and sequenced on 2× SP flow cells of Illumina NovaSeq 6000.