title: GSM8336613: Control sample-2; Homo sapiens; RNA-Seq
library_construction_protocol: In preparation for the RNA extraction, 50 mg of tissue was added to 500 μl TRIzol (Thermo Fisher Scientific) in which contained steel beads (the ratio of tissue, TRIzol and steel beads were 1:1:2~3). Samples were homogenized by using TissueLyser II (Qiagen, Hilden, Germany) for two cycles of two minutes at 20 Hz. After homogenization, Proteinase K (Worthington Biochemical Corp.) was added to remove protein contamination. For phase separation, the homogenized samples were added to BCP solution to separate into an interphase and upper aqueous phase. The upper aqueous phase was then carefully transferred into a fresh tube, and 100% ethanol was added for RNA precipitation. The procedures of RNA washing were performed using miRNeasy Mini Kit (Qiagen, Hilden, Germany), following the manufacturer's recommendations. The RNA was qualified by Fragment Analyzer 5200 (Agilent, Santa Clara, USA) and quantified by NanoDrop (Thermo Scientific, Waltham, MA, USA). The purified RNA was used for the preparation of the sequencing library by TruSeq RNA Exome Library Prep Kit (Illumina, San Diego, CA, USA) following the manufacturer's recommendations. Briefly, total RNA (120 ng) was fragmented into small pieces with divalent cation under elevated temperature. The first-strand cDNA was synthesized by reverse transcriptase and random primers. After the generation of double-strand cDNA and adenylation on 3' ends of blunt fragments, the adaptors were ligated and purified with AMPure XP system (Beckman Coulter, Beverly, USA). The purified libraries were hybridized with probes as biotinylated coding exome oligos. The streptavidin-coated magnetic beads were used to capture hybridized probes on the targeted regions of interest. The captured libraries were amplified by a second PCR. The quality of libraries was assessed on the Agilent Bioanalyzer 2100 system. The qualified libraries were then sequenced on Illumina NovaSeq 6000 platform with 150 bp paired-end reads generated by Genomics, BioSci & Tech Co., New Taipei City, Taiwan.