title: GSM9244525: ONEP subject 6 skin biopsy; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA was isolated from facial skin microbiopsy samples using TRIzol Reagent (Invitrogen Life Technologies) according to the manufacturer's instructions. RNA concentration, quality, and integrity were assessed using a NanoDrop spectrophotometer (Thermo Scientific) Sequencing libraries were constructed from 3 μg of total RNA per sample using the Illumina TruSeq Stranded mRNA LT Sample Prep Kit (or a similar proprietary Illumina protocol). Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. The purified mRNA was fragmented using divalent cations under elevated temperature in Illumina proprietary fragmentation buffer. First-strand cDNA was synthesized using random oligonucleotides and SuperScript II reverse transcriptase. Second-strand cDNA synthesis was performed using DNA Polymerase I and RNase H. The remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of the 3' ends of the DNA fragments, Illumina PE adapter oligonucleotides were ligated. cDNA fragments of 400-500 bp in length were size-selected and purified using the AMPure XP system (Beckman Coulter). DNA fragments with ligated adapters were enriched by 15 cycles of PCR using Illumina PCR Primer Cocktail. The final library quality and quantity were assessed using the Agilent High Sensitivity DNA assay on a Bioanalyzer 2100 system (Agilent Technologies)
identifiers.namespace: GEO
identifiers.id: GSM9244525_r1