GSM5610114: SKMEL5 1 uM binimetinib replicate 1; Homo sapiens; RNA-Seq
At the time of harvest, cells were washed with ice-cold 1X PBS over ice and lysed in TRIzol reagent (Thermo Fischer Scientific). Total RNA was isolated from each sample using Direct-zol RNA miniprep kit (Zymo Research) according to manufacturer's instructions. RNA were confirmed for quality using the Agilent Fragment Analyzer and 300ng of material was polyA selected using NEBNext® Poly(A) mRNA Magnetic Isolation Module (E7490) modified to include two rounds of polyA binding and 10 minute incubations. cDNA was generated using the NEB Ultra II directional kit (E7760) following manufacturers protocol using 12 cycles of PCR and an 0.9X SPRI clean. The resulting libraries were quality assessed using the Fragment Analyzer and quantified by qPCR prior to be sequenced on the Illumina HiSeq2000. The 50nt single-end reads with a depth of at least 10 million reads per sample were sequenced for all conditions.