RNA-Seq of MIA PaCa-2  in 3D spheroid: 19 hour treatment with trametinib
Two hundred nanograms of high purity RNA (RNA Integrity Number 7.0 or greater) was used as input to the TruSeq Stranded mRNA Library Prep Kit, High Throughput (Illumina), and the sample libraries were generated per manufacturers specifications on the Hamilton STAR robotics platform. The PCR amplified RNA-Seq library products were then quantified using the Fragment Analyzer Standard Sensitivity NGS Fragment Analysis Kit (Advanced Analytical Technologies). The samples were diluted to 10 nM in Elution Buffer (QIAGEN), denatured, and loaded at a range of 2.5 to 4.0 pM on an Illumina cBOT using the HiSeq 4000 PE Cluster Kit (Illumina). The RNA-Seq libraries were sequenced on a HiSeq 4000 at 75 base pair paired-end with 8 base pair dual indexes using the HiSeq 4000 SBS Kit, 150 cycles (Illumina). The sequence intensity files were generated on instrument using the Illumina Real Time Analysis software. The resulting intensity files were demultiplexed with the bcl2fastq2 software and aligned to the human transcriptome, quantified, and analyzed for differential expression using PISCES version 2017.09.02.