GSM7656532: Skin_HC_3; Homo sapiens; RNA-Seq
Human skin tissues were obtained as 4 mm punch biopsies or de-identified surgical tissue and stored in RNAlater at -80C before processing. To extract whole tissue RNA, samples were homogenized using a bead homogenizer and processed using the QIAGEN RNeasy Kit following the manufacturer's instructions. Following total RNA extraction, samples were treated with DNase (Turbo DNA-Free Kit, Invitrogen) following the manufacturer's instructions. Library preparation was performed with 1 ug of total RNA, and RNA integrity was determined using an Agilent Bioanalyzer. Ribosomal RNA was removed by a hybridization method using Ribo-ZERO kits (Illumina). mRNA was then fragmented in buffer containing 40 mM Tris acetate (pH 8.2), 100 mM potassium acetate, and 30 mM magnesium acetate and heated to 94C for 150 s. mRNA was reverse transcribed to yield cDNA using SuperScript III RT enzyme (Invitrogen, per manufacturer's instructions) and random hexamers. A second strand reaction was performed to yield ds-cDNA. cDNA was blunt ended, had an A base added to the 3 prime ends, and then had Illumina sequencing adapters ligated to the ends. Ligated fragments were then amplified for 12 cycles using primers incorporating unique index tags.