RNAseq of U2OS treated with HTH-01-015
Trizol RNA precipitations were preformed according to a modified manufacturers protocol: after the isopropanol precipitation step, the RNA pellet was resuspended in 100 l H2O 100 l acid phenol: chloroform (Ambion) was added, samples were vortexed for 20 seconds and spun at 13,000 rpm for 10 minutes. The aqueous phase was then extracted with 100 l of chloroform and then spun at 13,000 rpm for 3 minutes. The aqueous phase was then precipitated with 2 l glycogen, 10 l NaAc and 300 l EtOH and these were then stored at -20C for 24-48 hours. Pellets were washed with 500 l 75% EtOH, spun again at 13,000 rpm for 5 minutes. Pellets were allowed to air dry, then resuspended in 20 l H2O (milliQ). RNA concentrations were obtained using a NanoDrop One (Thermo Fisher Scientific).  RNA integrity was assessed using RNA ScreenTape (Agilent Technologies) according to the manufacturers protocol using a 2200 TapeStation System (Agilent Technologies). A RIN value > 7 was deemed acceptable. cDNA was generated using TruSeq stranded mRNA library prep kit (Illumina) followed by NextSeq500 High output 75 cycle sequencing.