title: GSM6863555: F21SCC_Visium (SCC samples from patients F21); Homo sapiens; RNA-Seq
library_construction_protocol: All samples were approved for research use under ethics approval numbers 2018000165 and 2017000318 by the University of Queensland's Human Research Ethics Committees and 11QPAH477 by the Metro South Human Research Ethics Committee. All formalin-fixed, paraffin-embedded (FFPE) blocks were previously prepared following a standard fixation procedure in 10% formalin, processed in ethanol and xylene and embedded in paraffin wax. The four melanoma samples (patients 6747-085P, 21031-08TB, 48974-2B, 66487-1A) were collected during 2008-2018 and all blocks were stored at room temperature. Full patient IDs are abbreviated as 6747, 21031, 48974 and 66487 throughout this manuscript.For BCC and SCC samples from eight patients (B18, E15, F21, P30, P13, P04, R01, D12), all fresh-shaved biopsies were obtained in accordance with the approved ethics protocol (11QPAH477). The patients presented to the Princess Alexandra Hospital Dermatology Department between October 2018 and February 2020. Among these patients, three patients (B18, E15, F21) were diagnosed with both SCC and BCC and biopsies of both cancer types were collected. Five patients (B18, P30, P13, P04, R01) kindly consented to participate in the collection of 4 mm punch biopsy samples of non-sun exposed healthy skin for paired scRNA sequencing experiments. Lesion identity was confirmed by pathological inspection. Portions of each sample from these patients were also preserved with 10% formalin as described for FFPE samples above. To process fresh samples for scRNA sequencing, briefly, fresh-shaved biopsies were collected in DMEM for immediate tissue dissociation. Tissue was incubated in 10 mg/mL Dispase II (cat. No. 04942078001, Roche, Darmstadt, Germany) for 45 min at 37°C, snipped into small pieces with scissors, and incubated in 0.25% Trypsin for 2 min. The cells were disrupted gently with a pipette and filtered through 70 µm and 40 µm cell strainers, taken up in culture medium and spun down at 350 rcf. Resuspended cells were collected in PBS containing Foetal Calf Serum for single-cell sequencing.     For single cell RNA sequencing, the RNA extraction followed the 10x Genomics Chromium single-cell library preparation and sequencing. For the spatial transcriptomics, with fresh frozen samples, the RNA was drawn from the tissue sections to the glass slide by permeabilization and gavity. The polyA-tail mRNA were captured by poly-T. For formalin fixed paraffin embedded samples, the probes hybridized to the targeted RNAs inside the tissue, and successfully ligated probes were extracted and captured on the glass slide. 10x Genomics Chromium single-cell library preparation and sequencing was performed according to the manufacturer's instructions, using the Single Cell 3' Library, Gel Bead and Multiplex Kit (version 2, PN-120233; 10x Genomics). Cell numbers were optimised to capture approximately 3,000 cells per reaction.For spatial transcriptomics, for fresh-frozen samples, tissue optimisation was performed as per the Visium Spatial Tissue Optimization User Guide (CG000238 Rev A, 10x Genomics), and a tissue permeabilisation time of 18-25 min was selected. For data generation, tissue was cryosectioned at 10 µm thickness and transferred to chilled Visium Spatial Gene Expression Slides (2000233, 10x Genomics, USA), and allowed to adhere by warming the back of the slide. Tissue sections were dried for 1 min at 37°C, fixed in chilled 100% methanol for 30 min, and stained with hematoxylin for 5 min and then eosin for 2 minutes, as per the Visium Spatial Gene Expression User Guide (CG000239 Rev A, 10x Genomics). Brightfield histology images were captured using a 10x objective on an Axio Z1 slide scanner (Zeiss). Brightfield images were exported as high-resolution tiff files using Zen software. This H&E staining and imaging protocol was used to stain all skin sections for histopathological annotation in this study. Libraries were sequenced on a NextSeq500 (Illumina) machine using a High Output 150 cycle kit (Illumina) at the University of Queensland Sequencing Facility, with the read structure as: Read1 - 28bp, Index1 - 10bp, Index2 - 10bp, Read2 - 120bp. The sequencing data was converted to FASTQ format as described above for scRNASeq data and the data was processed as described below. For FFPE samples, the FFPE blocks were sectioned at 5μm by rotary microtome and the sections were processed for spatial sequencing library prepration following the Visium Spatial Gene Expression for FFPE User Guide (CG000407, CG000408, CG000409 - 10x Genomics). The imaging and sequencing were similar to the protocol for the fresh-frozen samples as described above. Single-cell transcriptome libraries were sequenced on an Illumina NextSeq500 machine, using a 150-cycle High Output reagent kit (NextSeq500/550 version 2, FC-404-2002; Illumina) with the following reads and indices: read 2 (98 bp), i7 index (8 bp), read 1 (cell barcodes and UMI; 26 bp).