GSM7845289: WM4237-1, T1, treated, sample S1; Mus musculus; OTHER
OCT-embedded fresh frozen tissue was used. Five 10µm sections from each block were used for total RNA extraction (Qiagen), which was used for RNA integrity determination by RINe score using an Agilent Tapestation and High Sensitivity RNA ScreenTape. Blocks with RINe greater than 7 were optimized for ideal permeabilization time following the vendor protocol (10x Genomics, CG000238). Sections from four tissue blocks were placed on Visium Gene Expression slide for H&E staining and brightfield imaging via NanoZoomer SQ (Hamamatsu), block-specific tissue permeabilization, mRNA capture, and subsequent library generation per the manufacturer's protocol (10x Genomics, CG000239). Library concentration was quantified using a Tapestation High Sensitivity DNA ScreenTape (Agilent) and fluorometry (Thermofisher Qubit) and verified via KAPA qPCR. Libraries were pooled for sequencing on an Illumina NovaSeq 6000 using a 28-10-10-90 read configuration, targeting 100,000 read pairs per spot covered by tissue.