GSM5115022: spatial_transcriptomics_Reversal reaction 2; Homo sapiens; RNA-Seq
Skin samples were frozen in OCT medium and stored at -80°C until sectioning. Optimization of tissue permeabilization was performed on 20-μm-thick sections using Visium Spatial Tissue Optimization Reagents Kit (10X Genomics, Pleasanton, CA, USA), which established an optimal permeabilization time to be 6 minutes. Samples were mounted onto a Gene Expression slide (10X Genomics), fixed in ice-cold methanol, stained with hematoxylin and eosin, and scanned under a microscope (Keyence, Itasca, IL, USA). Tissue permeabilization was performed to release the poly-A mRNA for capture by the poly(dT) primers that are precoated on the slide and include an Illumina TruSeq Read, spatial barcode, and unique molecular identifier (UMI). Visium Spatial Gene Expression Reagent Kit (10X Genomics) was used for reverse transcription to produce spatially barcoded full-length cDNA and for second strand synthesis followed by denaturation to allow a transfer of the cDNA from the slide into a tube for amplification and library construction. Visium Spatial Single Cell 3ʹ Gene Expression libraries consisting of Illumina paired-end sequences flanked with P5/P7 were constructed after enzymatic fragmentation, size selection, end repair, A-tailing, adaptor ligation, and PCR. Dual Index Kit TT Set A (10X Genomics) was used to add unique i7 and i5 sample indexes and generate TruSeq Read 1 for sequencing the spatial barcode and UMI and TruSeq Read 2 for sequencing the cDNA insert, respectively.