GSM8727238: GeoMx ROI B12; Homo sapiens; OTHER
GeoMx: Formalin-fixed paraffin-embedded (FFPE) blocks were sectioned using a rotating microtome at 5 µm thickness and placed on standard, positively charged microscopy slides (TOMA). Slides were prepared for DSP according to the manufacturer's protocol. In brief, sections were deparaffinized and subjected to heat-induced protein epitope retrieval (using retrieval buffer at pH 9, Thermo Fisher Scientific, #00-4956-58) for 15 min at 100 °C using a steam cooker. Retrieval for mRNA was performed using proteinase K (Thermo Fisher Scientific, #25530049) at 1µg/ml for 15 min. Slides were then incubated with the human Whole Transcriptome Atlas (WTA) probe panel (Nanostring, #NA-GMX-RNA-NGS-HuWTA-4, Lot HWTA21003) at 37°C in a hybridization oven overnight. Sections were washed according to standard protocols and direct immunofluorescence was performed with a Melanoma morphology marker panel (Nanostring) composed of S100B, CD454 and a nuclear counterstain (SYTO-13). snRNA-Seq: Nucleus isolation with ST buffers was performed as per Slyper et al. 2020, and single nuclei were preapred using manufacturer's protocol (10X Genomics). GeoMx: Library was constructed using the manufacturer's protocol. The resulting library was sequenced by Novogene using an Illumina sequencer with the specifications provided by Nanostring (paired-end reads at length 27, index length 8) with 5% PhiX spike-in using a NovaSeq X Plus 25B PE150. snRNA-seq: Library was constructed using the 10X Genomics 3' v3.1 dual index manual, as per manufacturer's recommendations. The library was sequenced on Illumina NovaSeq X Plus 25B PE150 by Novogene, assuming approximately 50,000 reads per cell.