title: GSM9059914: hnscc_30.ROI_002.Tumor.budding; Homo sapiens; OTHER
library_construction_protocol: Slides were imaged in three fluorescence channels (FITC/525 nm, Cy3/568 nm, and Texas Red/615 nm) to visualize morphology markers, and regions of interest were segmented based on the expression of the morphology markers. For each tumor, two regions of interest (ROIs) were sampled from the bulk of the tumor and the surrounding stromal tissue. In budding cases, an additional two to three ROIs were sampled, focusing specifically on tumor bud cells and the adjacent stroma. Each ROI is then divided in distinct areas of illumination (AOIs) based on the morphology markers PanCK and CD45. PanCK-negative/CD45-positive cells were assigned the “immune” AOI,  PanCK-negative/CD45-negative cells were assigned the “stroma” AOI, and PanCK-positive/CD45-negative cells were assigned the “tumor” AOI. Each AOI was sequentially illuminated with UV and the UV-cleavable released tags were collected into 96-well plates. The library preparation was performed according to the GeoMx DSP NGS Readout User Manual. In short, the 96-well plate was dried by being left on the bench top overnight covered by a permeable membrane which was subsequently replaced by another permeable membrane and the plate was then spinned down. The samples were then rehydrated with ten µL nuclease-free water and the plate was pulse-centrifuged to 1000 x g. GeoMx Seq Code primers were used to amplify the WTA tags and add Illumina adaptor sequences and sample demultiplexing barcodes. The GeoMx Seq Code primer plate was thawed and the GeoMx Seq Code primer plate and the GeoMx NGS Master Mix were pulse-centrifuged to 1000 x g. The subsequent PCR reaction was set up on a PCR plate with two μL of GeoMx NGS Master Mix, four μL of GeoMx Seq Code primer mix (added to the corresponding PCR plate well), and four μL of DSP aspirate (added to the corresponding PCR plate well) used per well. The PCR plate was sealed with a PCR plate sticker, pulse centrifuged to 1000 x g, and incubated in the thermocycler with a 100°C heated lid. The cycling conditions were as follows: UDG treatment at 37°C for 30 min, followed by UDG deactivation at 50°C for ten min. Initial denaturation was performed at 95°C for three minutes. Amplification consisted of 18 cycles of denaturation at 95°C for 15 sec, annealing at 65°C for 60 sec, and extension at 68°C for 30 sec. A final extension was carried out at 68°C for five minutes, followed by an indefinite hold at 4°C. The PCR products were pooled using four μL of each PCR product (including the NTC) and the remainder of the NTC PCR product was placed into a separate tube. The tubes with the sample pool and the NTC went through the same AMPure cleanup protocol using the 1.2-fold volume AMPure XP beads and the libraries were sequenced on an Illumina NextSeq 550 with 27x27 paired end reads.