title: NextSeq 500 paired end sequencing; A robust experimental and computational framework analysis at multiple resolutions, modalities and coverages
design: A robust experimental and computational framework analysis at multiple resolutions, modalities and coverages
library_construction_protocol: Fresh shaved suspected SCC and BCC lesions and a 4mm punch biopsy of non-sun exposed skin from the same patient were collected in DMEM for immediate tissue dissociation. The tissue sections were rinsed in PBS and incubated in 8 mg/mL Dispase at 37°C for 1h, and were minced before a 3 minutes incubation in 0.25% Trypsin at 37°C. To collect single cells, the suspension was filtered through a 70µm cell strainer. Cells were collected in PBS containing Fetal Calf Serum.  Tissue cryosectioned at 10µm thickness were transferred to chilled Visium Tissue Optimization Slides (3000394, 10x Genomics, USA) and 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 one min at 37℃, fixed in chilled 100% methanol for 30 minutes and stained with hematoxylin and eosin for 5 minutes and 2 minutes as per Visium Spatial Tissue Optimization User Guide (CG000238 Rev A, 10x Genomics) or Visium Spatial Gene Expression User Guide (CG000239 Rev A, 10x Genomics) RNA extraction follows 10x protocol for single cell 3' sequencing and Visium FFPE sequencing protocols  The 10x Genomics Chromium scRNA-sequencing followed the manufacturer's instructions, using the Single Cell 3' Library, Gel Bead and Multiplex Kit (version 2, PN-120233; 10x Genomics). Cell numbers in each reaction were optimized to capture approximately 3,000 cells. Visium Spatial Gene Expression library was done following the manufacturer's protocol (CG000239 Rev A, 10x Genomics), using Visium V1, and the barcodes used are (B1:SI-TT-D5; C1: SI-TT-D6; D1: SI-TT-D7).