GSM5823107: HAP1 cell line HD1_141_3_S47275; Homo sapiens; RNA-Seq
Total RNA from knockout (KO) and inactive mutant expressing cells, as well as inhibitor-treated cells (JQ12 1 μM, MS-275 3 μM, both 24 hours) was isolated using Trizol reagent (Thermo Fisher Scientific) as described in the manual. The isolated RNA, dissolved in nuclease-free ddH2O, was then subjected to another precipitation step by adding 1/10 vol. 3 M sodium-acetate (pH 5.2) and 2.5 vol. 96% ethanol. Following overnight-incubation, the samples were centrifuged (12000 rpm, 30 min, 4 °C) and washed with 75% ethanol. Pellets were air-dried and dissolved in a suitable amount of nuclease-free ddH2O (shaking at 300 rpm, 10 min, 55 °C). RNA-seq libraries were prepared with the TruSeq Stranded mRNA LT sample preparation kit (Illumina) using Sciclone and Zephyr liquid handling robotics (PerkinElmer). Library amounts were quantified using the Qubit 2.0 Fluorometric Quantitation system (Life Technologies) and the size distribution was assessed using the Experion Automated Electrophoresis System (Bio-Rad). For sequencing, libraries were pooled, diluted and sequenced on an Illumina HiSeq 3000 instrument using 50 bp single-read chemistry.