GSM4792317: Control_Male_Treated; Mus musculus; RNA-Seq
Back skin was collected, and epidermis separated from dermis following incubation in a 1:1 dilution of dispase (Stem Cell Technologies, 07913) in Epilife media (Cascade Biologics, M-EPICF-500) at 37 °C for 1 hour. The epidermis was then lysed in TRIzol (ThermoFisher, 15596018), followed by chloroform extraction and RNA purification from the aqueous phase using ZYMO RESEARCH Quick-RNA MiniPrep per manufacturer's protocol. For RNA-seq, RNA was quantified using the NanoDrop ND-1000 spectrophotometer (ThermoFisher) and quality checked using the Agilent Bioanalyzer 2100 (Agilent). Library construction was performed according to the Illumina TruSeq® Stranded mRNA Sample Preparation Guide. One µg of total RNA was used and mRNA was enriched using oligo dT magnetic beads. The enriched mRNA was chemically fragmented for three minutes, followed by reverse transcription to make cDNA. The resulting cDNA was cleaned using AMPure XP beads, end repaired, and the 3' ends were adenylated. Illumina barcoded adapters were ligated on the ends and the adapter-ligated fragments were enriched by nine cycles of PCR. The resulting libraries were validated by qPCR and sized by Agilent Bioanalyzer DNA high sensitivity chip. The barcoded cDNA libraries were multiplexed on the Illumina HiSeq 4000 platform to yield 100-bp paired end reads