GSM8432328: PN lesional 4812; 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, Thermo Fisher Scientific, Waltham, Mass) in HBSS (Gibco, Thermo Fisher Scientific) at 4°C. The epidermis and dermis were separated. The epidermis was digested in 0.25% trypsin-EDTA (Gibco) with 10 U/mL DNase I (Thermo Fisher Scientific) for 1 hour at 37°C, quenched with FBS (R&D Systems [formerly Atlanta Biologicals], Bio-Techne, Minneapolis, Minn), and strained through a 70-μmol/L mesh. The dermis was minced, digested in 0.2% collagenase II (Life Technologies) and 0.2% collagenase V (Sigma-Aldrich, St Louis, Mo) in a plain medium for 1.5 hours at 37°C, and strained through a 70-μmol/L mesh. Epidermal and dermal cells were combined in a 1:1 ratio. The libraries were constructed by the University of Michigan Advanced Genomics Core on the 10x Chromium system with chemistry (version 3; 10x Genomics, Pleasanton, Calif). Libraries were then sequenced on the Illumina NovaSeq 6000 sequencer (Illumina Inc, San Diego, Calif) to generate 150-bp paired-end reads.