Illumina HiSeq 2500 paired end sequencing; RNA-seq of the human melanoma cell-line A375P treated with the BRAF inhibitor PLX4720 and a DMSO control
RNA-seq of the human melanoma cell-line A375P treated with the BRAF inhibitor PLX4720 and a DMSO control
A375-P cells treated with either 1 uM PLX4720 or 0.01% DMSO for 36 h. Cells were washed once with PBS, briefly incubated with trypsin at 37°C, re-suspended in ice-cold 2.5% FCS/PBS supplemented with either 1 uM PLX4720 or 0.01% DMSO and transferred into a 15 ml Falcon tube. Cells were counted, pelleted at 400 g for 4' at 4°C. Samples were filtered through a round-bottom test tube with a cell strainer snap cap (BD Falcon) to ensure single cell suspension. Sorts were performed on a BD FACSAriaIII or a BD FACSAriaIIIu Cell Sorter (BD Biosciences) using a 100 um nozzle.  200'000 cells were sorted directly into 2 ml Eppendorf tubes containing 700 L RA1 (lysis buffer) from the NucleoSpin® RNA II RNA isolation kit (Macherey-Nagel) containing 70 l TECP (Sigma-Aldrich). Eppendorfs were vortexed regularly to ensure cell lysis. RNA was isolated according to the manufacturer's protocol. The integrity of the RNA samples was checked using the Bioanalyzer system. Their concentration was measured using Quant-IT RiboGreen RNA Assays (Life Technologies). Libraries for Illumina sequencing have been prepared with 200 ng total RNA input material using the TruSeq Stranded Total RNA Library Prep kit Ribozero (Illumina) and quality checked using the Fragment Analyzer standard sensitivity NGS kit (AATI).
Experimental Factor: compound
PLX4720
Experimental Factor: dose
1