title: Sample 5
organism: Homo sapiens
description: Protocols: ex-vivo skin samples with variable skin color were used for the study. Normal human skin samples were obtained after breast reduction surgery from healthy women living in France  Epidermis was separated from the dermis using NaBr (1h at 37°C), and RNA was isolated from the epidermis. Total RNA was isolated from the 21 samples with the RNeasy Mini kit (Qiagen, Germantown, MD, USA). Purity and integrity of the RNA was assessed on the Agilent 2100 Bioanalyzer with the RNA 6000 NanoLabChip reagent set (Agilent, Palo Alto, CA, USA) and the Nanodrop spectrophotometer. Library preparation and RNA sequencing Library preparation and RNA-seq were carried out as described in the Illumina TruSeq stranded mRNA User Guide (Illumina inc., San Diego, CA, USA). Briefly, the first step in the workflow involved purifying the poly-A containing mRNA molecules from 600 ng of total RNA using poly-T oligos attached magnetic beads. Following purification, the mRNA was fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase and random primers. The cDNA fragments were then purified with Agencourt AMPure XP beads (Beckman Coulter, Missisauga, Ontario, Canada) and enriched with 13 cycles PCR to create the final cDNA library. cDNA quality was assessed on the TapeStation 2200 (Agilent technologies, Santa Clara, CA, USA) and the library was quantified using the Qbit 3.0 fluorimeter (ThermoFischer Scientific, Canada).