title: GSM9076581: Patient 2_ROI 09_ VIM+; Homo sapiens; OTHER
library_name: GSM9076581
library_construction_protocol: GeoMx™ digital spatial profiling was performed as per the supplier's protocol using the human Whole Transcriptome Atlas probe set with read-out by next-generation sequencing (Nanostring Technologies). Briefly, 5 µm FFPE sections of a sporadic DICER1-thyroid case and a germline DGCR8- thyroid case, were processed through deparaffinization, antigen retrieval (Tris-EDTA ph9 for 20 min at 98°C), and Proteinase K digestion (1 ug/mL Proteinase K for 15 min at 37°C) prior to overnight incubation at 37°C with the human Whole Transcriptome Atlas diluted in hybridization Buffer R. The next day, slides were washed twice in 2XSSC/formamide (25 min at 37°C), blocked with Buffer W (30 min at RT) and incubated with an antibody cocktail consisting of PanCK-AF532, CD45-AF594, VIM-AF647, and SYTO13 for 1 hr in a humidity chamber. Slides were washed 3 times in 2XSSC and then scanned on the GeoMx™ DSP instrument (at 20X), after which regions-of-interest (ROIs) in each case were laid down to capture different histological components in triplicates at least. For the DICER1-case, this included a tumour core from a DICER1-mutated PDTC, the tumor's capsule, a non-neoplastic nodular area (DICER1-wildtype benign thyroid lesion) and normal area (DICER1-wildtype). For the DGCR8-case, this included a tumour core from a DGCR8-mutated microPTC, a normal adjacent area, non-neoplastic nodular areas (DGCR8-mutated benign thyroid lesions) and normal area (DGCR8-mutated). A total of 51 ROIs were selected and were segmented further into PanCK+ and/or VIM+ areas, and Whole Transcriptome barcodes were released from these distinct populations by UV light illumination and microfluidic collection. The UV-released barcodes from each of the 84 area-of-illuminations (AOIs) were PCR-amplified to add unique dual-index Illumina i5 and i7 indices and bead-purified twice using AMPure XP beads (Beckman Coutler, A63881). Pooled libraries were sequenced on two 75-cycle High Output flowcells on an Illumina NextSeq sequencer, generating 916M unique raw reads. FASTQ files were converted to DCC files and uploaded onto the GeoMx™ DSP for trimming, stitching, aligning, and deduplication.