GSM5390621: E733-S: Patient 99 HTS; Homo sapiens; RNA-Seq
RNA was isolated from 108 human samples comprising of uninjured skin (n=26), acute burn wounds (n=54), and hypertrophic scars (HTS) (n=30). Frozen tissue was pulverized using mortar and pestle in liquid nitrogen to maintain temperature during processing. Total RNA, including small RNAs such as microRNAs, non-coding RNA, messenger RNA, and ribosomal RNA was isolated using the MagMAX™ mirVana™ Total RNA Isolation Kit (Applied Biosystems) followed by DNAse digestion on Purelink RNA mini kit spin columns (Thermo Fisher Scientific). Total RNA is normalized to 7.5ng/ul in a total volume of 50ul on the Perkin Elmer Janus Workstation (Perkin Elmer, Janus II). Poly-A selection and cDNA synthesis are performed using the TruSeq Stranded mRNA kit as outlined by the manufacturer (Illumina, cat#RS-122-2103). All steps are automated on the Perkin Elmer Sciclone NGSx Workstation to reduce batch to batch variability and to increase sample throughput. Final RNASeq libraries are quantified using the Quant-it dsDNA High Sensitivity assay, and library insert size distribution is checked using a fragment analyzer (Advanced Analytical; kit ID DNF474). Samples where adapter dimers constitute more than 4% of the electropherogram area are failed prior to sequencing. Technical controls (K562,Thermo Fisher Scientific, cat# AM7832), are compared to expected results to ensure that batch to batch variability is minimized. Successful libraries are normalized to 10nM for submission to sequencing.