GSM9096549: Skin9, Normal, biol rep4; Homo sapiens; RNA-Seq
20-30mg frozen tissue was homogenized in 1mL TRIzol Reagent (ThermoFisher catalog # 15596018) using the QIAGEN TissueLyser at 15Hz for 2-3 minutes with a Stainless Steel Bead (QIAGEN catalog # 69989). Phase separation was induced with 200 µL chloroform. RNA was precipitated from the aqueous phase with isopropanol and linear acrylamide and washed with 75% ethanol. The samples were resuspended in 13 µL nuclease-free water. After RiboGreen quantification and quality control by Agilent BioAnalyzer, 0.7-1 µg of total RNA with DV200 percentages varying from 70-88% underwent ribosomal depletion and library preparation using the TruSeq Stranded Total RNA LT Kit (Illumina catalog # RS-122-1202) according to instructions provided by the manufacturer with 8 cycles of PCR. Samples were barcoded and run on a HiSeq 4000 in a PE50 run, using the HiSeq 3000/4000 SBS Kit (Illumina). On average, 46 million paired reads were generated per sample and 26% of the data mapped to the transcriptome.