GSM3762950: Mutant RAF1 S257L/+ PD98059 rep1; Homo sapiens; RNA-Seq
Total RNA was prepared using the mirVana PARIS purification Kit (ThermoFisher, #AM1556) following the manufacturer's recommendations. RNA integrity (RIN) was determined using ScreenTape R6K kit on the TapeStation2200 (Agilent Technologies) and only samples with RIN >7 were further used for RNA-sequencing Isolated total RNA was diluted to 0.95 ng/µl and 10.5 µL was loaded in each reverse transcription reaction with the SuperScript VILO cDNA Synthesis Kit (ThermoFisher, #11754050). Reverse transcription was completed in a thermocycler with the following temperature settings: 42ºC for 30 minutes, 85ºC for 5 minutes and 10ºC on hold. After reverse transcription, libraries were prepared using the Ion Chef (ThermoFisher, #4484177) and the Ion AmpliSeq Transcriptome Human Gene Expression Panel, Chef-Ready Kit (ThermoFisher, #A31446). The AmpliSeq Gene Expression Panel includes one primer pair for every gene in the transcriptome, allowing for amplification of a single amplicon for each gene to be sequenced. At the completion of library preparation, libraries from 8 samples were pooled together, templated, and loaded on the Ion 540 Chip (ThermoFisher, #A27765) using the Ion Chef and the Ion 540 Kit Chef (ThermoFisher, #A27759). After templated samples were loaded onto the 540 Chips, each Chip was inserted into the Ion Torrent S5 sequencer (ThermoFisher, #A27212) and 500 flows were applied for each sequencing run. The second Chip was stored at 4ºC during the first sequencing run and removed to room temperature for 30 minutes prior to running the second Chip