title: Fine mapping of cell states across anatomical sites in healthy human skin and basal cell carcinoma -  Spatial Transcriptomics - 10X Visium
design: Fine mapping of cell states across anatomical sites in healthy human skin and basal cell carcinoma -  Spatial Transcriptomics - 10X Visium
library_construction_protocol: Fresh frozen OCT-embedded skin biopsies were cryosectioned as 10 μm sections, placed onto SuperFrost Plus glass slides (Thermo Fisher Scientific, Waltham, MA, USA) and stored for less than a week at -80˚C prior to high sensitivity library preparation. Optimal RNA integrity on the skin sections was assessed by RNAscope using three housekeeping genes with high (UBC) and medium (PPIB) expressors (Supplementary figure 3B). We also validated the integrity of the Visium library after sequencing: all samples presented good RNA quality (Supplementary figure 3C). However, we excluded 2 of the 32 10X Visium samples from further analysis due to poor tissue attachment to the slide.  Visium spatial gene expression slides and reagents were used according to manufacturer instructions (10X Genomics). Each capture area (6.5 x 6.5 mm2) contains 5,000 barcoded spots that were 55 μm in diameter (100 μm center to center between spots) providing an average resolution of 1 to 10 cells up to a maximum of 100 cells depending on tissue area. Fresh frozen skin samples were embedded in OCT, cryo-sectioned and processed according to the recommended protocols (Tissue optimization: CG000238; Gene expression: CG000239). Optimal permeabilization time for 10um skin sections was 20minutes. cDNA libraries were quality controlled using the Agilent Bioanalyser. The cDNA libraries were sequenced on the Illumina HiSeq 4000 system, targeting 300 million reads per section with parameters 28cy R1, 8cy i7 index, 0cy i5 index, 91cy read 2.