RNA-seq of mouse keratinocytes treated with siRNA against embigin
RNA-seq of mouse keratinocytes treated with siRNA against embigin
Mouse keratinocytes isolated from adult dorsal skin were cultured in the comprised FAD medium: three parts DMEM medium (Lonza), one part Ham's F12 medium (Thermo Fisher Scientific) supplemented with 10% FCS (Biowest), 2 mM L-glutamine (Lonza), 10 units/ml Pen-Strep (Lonza), 200 µM adenine (Sigma-Aldrich), 0.5μg/ml hydrocortisone (Sigma-Aldrich), 5μg/ml insulin (Sigma-Aldrich), 16,8 ng/ml cholera toxin (Sigma-Aldrich) and 10 ng/ml epidermal growth factor (Sigma-Aldrich. The cells were cultured at 37°C in a humidified atmosphere with 5% CO2. Subconfluent mouse keratinocytes were transfected with 75 nM siRNA (Mm_Emb_1 and Mm_Emb_4 Flexitube siRNAs and AllStars Negative Control siRNA from QIAGEN) by using siLentFect reagent (Bio-Rad) according to manufacturer's instructions and cultured for 48 h. Total RNA was isolated by using the NucleoSpin RNA kit (Macherey-Nagel) according to the manufacturer's instructions. Library preparation was performed according to TruSeq Stranded mRNA Sample Preparation protocol (Illumina, 15031047)and TruSeq Stranded mRNA HT Kit (Illumina)