RNAseq of MDA231
RNA library preparation was performed as described in the Illumina TruSeq Stranded Total RNA LT sample preparation kit with Ribo-Zero Gold (Illumina, Inc.). In brief, ribosomal RNA was depleted from 0.3-1 g of total RNA. Following depletion, mRNA was fragmented to an average size of 200-400 bp at 94C for 4 min. The cleaved RNA fragments were copied into first-strand cDNA using a reverse transcriptase (Invitrogen) and random primers. The first-strand cDNA was converted into double-stranded DNA in the presence of dUTP. The incorporation of dUTP in second-strand synthesis quenched the second strand during amplification and thus improved the strand specificity of the library. These cDNA fragments then had the addition of a single 'A' base and subsequent ligation of the adapter. The products were purified and enriched with PCR to create the final library. After qualification with the Agilent 2100 Bioanalyzer (Agilent Technologies) with the DNA chip and quantification with the Qubit 3.0 Fluorometer (Invitrogen), the samples were subjected to 150-bp paired-end sequencing using an Illumina HiSeq X TEN platform (Illumina, Inc.).