GSM5492939: Full thickness skin - RDEB2 epidermal grafted site; Homo sapiens; RNA-Seq
Full-thickness 4-mm punch biopsies were obtained from RDEB patients or a healthy donor. Samples were kept on ice in DMEM with 1% gentamycin for no longer than one hour before their enzymatic and mechanical dissociation with the Whole Skin Dissociation kit for human material (Miltenyi Biotec, cat. no. 130-101-540) and the gentleMACS dissociator (Miltenyi Biotec), following the manufacturer's instructions. Cell suspensions were then filtered through 70-μm cell strainers (Falcon). Full-thickness skin biopsies were incubated in enzyme mix for 8 hours, mechanically dissociated using Miltenyi gentleMACs and dead cells removed using Stem Cell Technologies EasySepTM Dead cell Removal (Annexin V) kit (catalog #17899) to remove dead cells and debris. Epidermal grafts were dissociated using Miltenyi Epidermal Dissociation Kit (#130-103-464), incubated in enzyme mix for one hour (due to decreased thickness of tissue) and then dead cells removed using Stem Cell Technologies kit as described above. Viability was between 30-60% for all samples. Samples were resuspended at 10,000 cells/mL in cold PB buffer (phosphate-buffered saline with 0.5% bovine serum albumin). Samples were kept on ice for immediate transport to the University of Minnesota Genome Core for library preparation. Cells were subjected to 10X Genomics Chromium Single-Cell Platform manipulation (3' library prep), followed by sequencing using Illumina NovaSeq 6000 system, and then results were run through Cell Ranger pipeline software for sequence alignment and basic filtering. Sequencing libraries were subsequently prepared following the Drop-seq methodology, using a Chromium Single Cell Controller and the v3 chemistry from 10X Genomics (cat. no. PN-1000075).