RNA-seq of normal human keratinocytes (NHEK) treated with tapinarof against untreated control
RNA-seq of normal human keratinocytes (NHEK) treated with tapinarof against untreated control
Normal human epidermal keratinocyte (NHEK) (Cat. C-12003, Promocell) vials from different donors were obtained and cultured in Dermacult medium (Cat. 100-0500, Stemcell Technologies) containing Keratinocyte Expansion Supplement (Cat. 100-0500, Stemcell Technologies), Hydrocortisone (Cat. 07925, Stemcell Technologies), penicillin (50 U/ml) and streptomycin (50 μg/ml) (Cat. 4-01F00-H, BioConcept). Keratinocytes were cultured in T75 flasks and medium was exchanged every other day. After reaching 90% confluency, keratinocytes were washed with D-PBS (Cat. 37354, Stemcell Technologies) and treated with ACCUTASE (Cat. 07922, Stemcell Technologies). After 5-7 minutes at 37°C, keratinocyte were dislodged with gentle tapping of the plate, transferred to a 15ml conical tube and washed with the keratinocyte medium. For the treatment, keratinocytes were plated into a 12 well plate at a density of 1x 105 cells per well and cultured for 24h in keratinocyte medium. After 24h, the medium was exchanged to keratinocyte medium containing 1.2mM CaCl2 (Cat. C34006, Sigma-Aldrich) and NHEKs were treated with DMSO or tapinarof (1 μM. at. #S9700, Selleck Chemicals). NHEKs were harvested after 3 days of treatment The RNeasy Micro Kit (Cat. 74004, Qiagen) was used for total RNA isolation according to manufacturer's instructions  Illumina TruSeq Stranded mRNA protocol (Directional, first strand)