GSM7932788: SCD-925YJ1; Homo sapiens; RNA-Seq
Generation of single-cell suspensions for scRNA-seq was performed as follows: Skin biopsies were incubated overnight in 0.4% dispase (Life Technologies) in Hank's Balanced Saline Solution (Gibco) at 4°C. Epidermis and dermis were separated. Epidermis was digested in 0.25% Trypsin-EDTA (Gibco) with 10U/mL DNase I (Thermo Scientific) for 1 hour at 37°C, quenched with FBS (Atlanta Biologicals), and strained through a 70μM mesh. Dermis was minced, digested in 0.2% Collagenase II (Life Technologies) and 0.2% Collagenase V (Sigma) in plain medium for 1.5 hours at 37°C and strained through a 70μM mesh. For spatial-seq, skin samples were frozen in OCT medium and stored at -80°C until sectioning. Optimization of tissue permeabilization was performed on 20 μm sections using Visium Spatial Tissue Optimization Reagents Kit (10X Genomics, Pleasanton, CA, USA), which established an optimal permeabilization time to be 9 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). For single cell RNA-seq, for the samples collected from the University of Pittsburgh, epidermal and dermal cells were combined in 1:1 ratio. For three of the samples collected from the University of Michigan, the epidermal and dermal cells were prepared in different libraries that were constructed by the University of Michigan Advanced Genomics Core on the 10X Chromium system with chemistry v3.  For the remaining samples the epidermis and dermis were combined in 1:1 ratio.  Libraries were then sequenced on the Illumina NovaSeq 6000 sequencer to generate 150 bp paired-end reads. For spatial-seq, 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. Libraries were then sequenced on the Illumina NovaSeq 6000 sequencer to generate 150 bp paired end reads.