Figure 1.

Experimental design of the mixture control experiment. RNA from two lung cancer cell lines (NCI-H1975 and HCC827) were obtained after culture on three separate occasions to obtain samples for three replicates to simulate some degree of biological variability. RNA from each replicate was either kept pure or mixed in three different proportions. The second replicate of each mixture was split in two and either processed normally or heat treated (incubated at 37°C for 9 days, see Materials and Methods) to degrade the RNA and simulate variations in sample quality. Each sample was then processed using either Illumina's TruSeq RNA v2 kit (Poly-A mRNA) or Illumina's TruSeq Total Stranded RNA kit with Ribozero depletion followed by sequencing on an Illumina HiSeq 2500 to obtain 100 bp single-end reads for further analysis.