title: GSM5348322: hdSEs (G2_2); Homo sapiens; RNA-Seq
library_construction_protocol: The dermis of two individual hdSEs at day 23 was separated from the epidermis with forceps and subsequently cut into small pieces and incubated in 1 ml of TDB containing 250 μg Liberase TL for 45 min at 37 °C. To isolate NHEK the epidermis was incubated in 3 ml of PBS- with gentle agitation. PBS- was aspirated, the epidermis cut into small pieces and 1 ml of 0.05 % Trypsin/EDTA were added for 15 min at 37 °C. 100 μl of FCS were added and NHEK were released by thorough resuspension. Cells were collected by centrifugation at 300 g for 5 min, resuspended in 500 μl INF medium containing 0.5 μg DAPI and filtered through a 35 μm cell strainer. Viable NHDF and NHEK were sorted based on their negative DAPI signal with a FACSAria III cytometer (BD Biosciences) and collected in a single tube. Cell density was evaluated by microscopy using a hemocytometer and adjusted to 1000 cells/μl. Subsequently, the Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 (10x Genomics) were used for reverse transcription, cDNA amplification, and library construction according to the manufacturers' instructions. 16.5 μl of cell suspension were used to recover 10,000 cells and libraries were quantified with a Qubit 3 Fluorometer (Thermo Fisher Scientific). Quality was checked using a 2100 Bioanalyzer with High Sensitivity DNA kit (Agilent). Sequencing was performed in paired-end mode with a SP flow cell (2 x 50 cycles) using NovaSeq 6000 sequencer (Illumina).