title: GSM5508640: M17D0AR_2; Homo sapiens; RNA-Seq
library_construction_protocol: Cultured cells were harvested then centrifuged at 1,500 g and cell pellets were used for RNA isolation. Cell pellet was homogenized in TRIzol using a hand-held pestle homogenizer and incubated in TRIzol for at least 5 minutes. Chloroform (1:5 ratio) was added, mixed well and incubated at room temperature for 15 minutes. Samples were then centrifuged at 12,000 g for 15 minutes at 4°C. The top aqueous layer was transferred to a clean tube, and the RNA was precipitated in 3 M NaAc pH 5.2 (10:1 ratio), 4 ml of glycogen (5 mg/ml), 100% isopropanol (1:1 ratio) overnight at -80°C. The next day, the samples were centrifuged at 20,000 g for 20 minutes at 4°C. The resulting RNA pellet was washed once in 75% ethanol, centrifuged at 7,500 g for 10 minutes at 4°C. The washed RNA pellet was dissolved in nuclease-free water. RNA was quantified by NanoDrop and the quality was confirmed by agarose gel. For rRNA-depleted RNA-seq library, sample quality was assessed by Bioanalyzer 2100 Eukaryote Total RNA Pico (Agilent Technologies, CA, USA) and quantified by Qubit RNA HS assay (ThermoFisher). Ribosomal RNA depletion was performed with Ribo-zero rRNA Removal Kit (Illumina Inc., San Diego, CA) followed by NEBNext® Ultra™ II Nondirectional RNA Library Prep Kit for Illumina® per manufacturer's recommendation. Library concentration was measured by qPCR and library quality evaluated by Tapestation High Sensitivity D1000 ScreenTapes (Agilent Technologies, CA, USA). Equimolar pooling of libraries was performed based on qPCR values. Libraries were sequenced on a HiSeq with a read length configuration of 150 PE targeting 80M total reads per sample (40M each direction).