GSM7622799: Skin_Donor A (40Y)_Single-hair region_Sample A5; Homo sapiens; RNA-Seq
To generate single-cell suspensions from human skin samples, the following four experimental steps were performed: Firstly, skin tissues were dissected into small pieces containing only a single hair (single-hair samples) or no hairs (non-hair samples); Secondly, to maximize the number of dermal cells for analysis, the interfollicular epidermis was removed from each sample using tweezers after treating skin tissues with 2.4 U/mL dispase (Corning) in PBS at 4ºC for 20 minutes and then at 37ºC for 20 minutes. In this process, care was taken not to remove hair follicle keratinocytes; Thirdly, remaining skin tissues were placed in a mixture of the following enzymes: 0.20% collagenase I (Thermo Fisher Scientific), 0.05% collagenase II (Thermo Fisher Scientific), 0.05% collagenase IV (Thermo Fisher Scientific), 0.01% Hyaluronidase (Nakalai Tesque), and 5 U/mL DNase I (Sigma) in HBSS. The skin tissue was minced using surgical scissors and incubated at 37ºC with gentle rotation (20 rpm) until the tissue was completely digested (for about one hour). Finally, a skin cell suspension was obtained from each sample. The obtained cell suspension was passed through 70- and 30-µL cell strainers (Miltenyi Biotec) and centrifuged at 3000 rpm for seven minutes. After removal of the supernatant, cells were resuspended with FBS-containing DMEM. For each skin tissue sample, a single-cell cDNA library was individually prepared using Single Cell 3' Reagent Kit of version 3 chemistry (10x Genomics) following the manufacturer's protocol. The prepared cDNA libraries were then sequenced using NovaSeq 6000 Sequencing System (Illumina).