GSM5150552: COLO205_4_hr_selumetinib; Homo sapiens; RNA-Seq
Cell pellets were lysed in 1 mL TRI reagent (Sigma, T9424) and mixed until a homogeneous lysate was obtained. For phase separation, 0.2 mL chloroform was added, mixed thoroughly and allowed to stand for 10 minutes prior to centrifugation at 12,000 × g for 15 min at 4 °C. The upper aqueous phase containing RNA was transferred to a fresh tube, and 0.5 mL 2-propanol per mL original lysate was then added, samples mixed and allowed to stand for 10 minutes. Samples were centrifuged (12,000 × g for 10 min at 4 °C), and RNA pellets washed with 1 mL 75% (v/v) ethanol (7,500 × g for 5 min at 4 °C). Pellets were air-dried for 5-10 minutes and resuspended in RNase-free water. RNA prepared from flow sorted cells was quality controlled on a Bioanalyzer 2100 (Agilent) using RNA Pico 6000 chips (Agilent 5067-1513). mRNA-seq libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB E7760S) with the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB E7490S), following the protocol provided by the manufacturer with the exception that two successive 0.9x AMPure purifications were performed on the final amplified libraries. All libraries were amplified using 12-14 PCR cycles. Library quality was assessed on a Bioanalyzer 2100 using High Sensitivity DNA chips (Agilent 5067-4626) and quantification performed with a KAPA Library Quantification Kit (Roche, KK4844). Libraries were sequenced by the Babraham Institute Next Generation Sequencing Facility on an Illumina HiSeq2500 in Rapid Run 50bp Single End mode.