title: Visium Spatial data of Brain section from Parkinson Mouse Model based on inducible expression of human a-syn constructs
library_construction_protocol: To prepare tissue samples for Visium Spatial Gene Expression experiments, fresh-frozen tissue specimens were utilized. The animals were euthanized using fast cervical dislocation and the brain was immediately dissected. A metal beaker was filled with isopentane and placed into a liquid nitrogen dewar to allow for rapid cooling. Sufficient contact between the isopentane and the liquid nitrogen was ensured. The fresh tissue sample was positioned in a cryomold and coated with TissueTek embedding medium. The cryomold containing the embedded tissue was lowered into the chilled isopentane and held in place until the TissueTek medium had fully solidified. Once the tissue was completely frozen, it was transferred to dry ice and afterwards stored in a sealed container at -80 °C for long-term preservation. For sectioning, the cryostat was set to -20 °C. The frozen tissue block was removed from storage and allowed to equilibrate briefly at cryostat temperature. The tissue block was sectioned into slices with a thickness of 10-12 µm using the cryostat. Each section was carefully transferred onto a Visium Gene Expression slide by gently touching the section to the active surface of the slide. After mounting the tissue section, the slide was immediately placed into a storage container at -80 °C. The slides were maintained at this temperature until further processing to preserve RNA integrity and spatial organization. The experiments following fluorescence staining were carried out using the “Visium Spatial Gene Expression Slide & Reagent Kit” (10X Genomics) in accordance with the manufacturer's protocol. In summary, the procedure involved several key steps to ensure successful library preparation and gene expression analysis. Initially, tissue sections were subjected to controlled permeabilization to allow access to intracellular RNA. This was followed by reverse transcription. Next, second strand synthesis was performed to generate double-stranded cDNA. The resulting cDNA was denatured to separate the strands, and the single-stranded cDNA was subsequently amplified through a series of PCR cycles. The amplified cDNA underwent a cleanup process to remove reaction by-products. Following cDNA purification, the samples were subjected to a step of fragmentation, end repair and A-tailing to create suitable 3' ends for adaptor ligation. Adaptors were then ligated to the DNA fragments to facilitate downstream sequencing. Post-ligation cleanup was performed to eliminate excess adapters. Finally, a sample index PCR was carried out to introduce unique sample indices, enabling the multiplexing of samples during sequencing. The experiments following fluorescence staining were carried out using the “Visium Spatial Gene Expression Slide & Reagent Kit” (10X Genomics) in accordance with the manufacturer's protocol. In summary, the procedure involved several key steps to ensure successful library preparation and gene expression analysis. Initially, tissue sections were subjected to controlled permeabilization to allow access to intracellular RNA. This was followed by reverse transcription. Next, second strand synthesis was performed to generate double-stranded cDNA. The resulting cDNA was denatured to separate the strands, and the single-stranded cDNA was subsequently amplified through a series of PCR cycles. The amplified cDNA underwent a cleanup process to remove reaction by-products. Following cDNA purification, the samples were subjected to a step of fragmentation, end repair and A-tailing to create suitable 3' ends for adaptor ligation. Adaptors were then ligated to the DNA fragments to facilitate downstream sequencing. Post-ligation cleanup was performed to eliminate excess adapters. Finally, a sample index PCR was carried out to introduce unique sample indices, enabling the multiplexing of samples during sequencing.