title: RNAseq of A375 melanoma cell line
design: Total RNA was isolated using the RNeasy Plus Mini Kit (QIAGEN) and quantified using RNA 6000 Nano Kit (Agilent Technologies) on Agilent 2100 BioAnalyzer. 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 manufacturer's specifications on Hamilton STAR robotics platform. PCR-amplified RNA-Seq library products were then quantified with Fragment Analyzer Standard Sensitivity NGS Fragment Analysis Kit (Advanced Analytical Technologies). Samples were diluted to 10 nmol/L in Elution Buffer (QIAGEN), denatured, and loaded at a range of 2.5 to 4.0 pmol/L on an Illumina cBOT using HiSeq 4000 PE Cluster Kit (Illumina). RNA-Seq libraries were sequenced on a HiSeq 4000 at 75 base pair paired-end with 8 base pair dual indexes using HiSeq 4000 SBS Kit, 150 cycles (Illumina). Sequence intensity files were generated on instrument using Illumina Real Time Analysis software. Resulting intensity files were demultiplexed with the bcl2fastq2.