Visium spatial gene expression slide and reagent kit

Protocols for Visium tissue optimization and spatial gene expression were conducted as described by 10x Genomics (https://10xgenomics.com/) using H&E as the counterstain. Briefly, Visium spatial gene expression was processed using a Visium spatial gene expression slide and reagent kit (10x Genomics, PN-1000184). For each well, a slide cassette was used to create leakproof wells for adding reagents. Then, 70 μL permeabilization enzyme was added and incubated at 37°C for 12 min to achieve optimal permeabilization. Each well was washed with 100 μL of SSC, and 75 μL of reverse transcription Master Mix was added for cDNA synthesis.

Visium spatial gene expression slide and Reagent Kit (10× Genomics, PN-1000184) was used to detect spatial gene expression. Slides were inserted into slide cassettes that created leakproof wells for permeabilization with 70 μL enzyme, and incubated at 37 °C for 18 min. Each well was washed with 100 μL SSC, and added 75 μL reverse transcription (RT) Master Mix for cDNA synthesis.

Tissue slices were processed according to the standard Deparaffinization and H&E Staining and Decrosslinking protocol (10x Genomics, CG000409). Briefly, slices were deparaffinized and stained with hematoxylin, followed by bluing and eosin staining. Slices were covered with mounting medium (85% glycerol) and coverslipped before imaging. Slides were imaged using the Pannoramic SCAN slide scanner (3D Histech) controlled by the Pannoramic Scanner Software. Straight after imaging, coverslips were removed and samples were directly processed according to the standard Visium Spatial Gene Expression protocol (10x Genomics, CG000407) using the Visium Spatial Gene Expression Slide and Reagent Kit (10x Genomics, PN-1000185). Decross-linking was then performed, and probes were immediately hybridized, ligated, released, and collected for library construction following manufacturer’s recommendations (10x Genomics, CG000409). Tissue-covered spots were quantified, and libraries were pooled according to their concentration and spot occupation on slides. Library pool was sequenced using NovaSeq 6000 (Illumina) using the recommended parameters: read 1, 28 cycles; i7 index, 10 cycles; i5 index, 10 cycles; and read 2, 50 cycles.