GSM6892084: BF_LE_03_VC_03_all; Homo sapiens; RNA-Seq
Cell suspensions were generated using the Whole Skin Dissociation Kit (Miltenyi®, Bergisch Gladbach, Germany) according to the manufacturer's specifications. Single-cell suspensions were subsequently counted for the number of dead cells by Trypan blue staining (Sigma Aldrich, Munich) which did not exceed 5% of cells. Single cells were loaded on a 10x Genomics®) Chromium Next GEM Chip G (10x Genomics®, Pleasanton, CA, U.S.A.) according to the manufacturer's specifications. Overall, 10,000 cells were loaded into one channel of the Chromium system using the v3.1 single-cell reagent kits (10x Genomics®). Following capture and lysis, cDNA was synthesized and amplified for 12 PCR cycles. The amplified cDNA was then used to construct Illumina®) sequencing libraries according to the instruction of the Chromium User Guide® (10x Genomics®). Briefly, blunt end repair was followed by adapter ligation. During PCR amplification, sample indices were added. The barcoded libraries were purified with SPRI select beads and quantified using Qubit®) dsDNA HS Assay Kit (Invitrogen, Thermo Fisher Scientific, Waltham, Massachusetts, USA) according to the manufacturer's instructions. Correct size distribution of the library DNA was analyzed using the Fragment Analyzer (Agilent®), Santa Clara, CA, U.S.A.). Sequencing of 2x150 bp was performed with an Illumina®) NextSeq 550 sequencer (Illumina®), San Diego, CA, USA). Demultiplexing was done using bcl2fastq v2.20.0.422 (Illumina®).