title: Transcriptomics of Kidney Cancer
design: RNA sequencing libraries were prepared using the Illumina TruSeq Stranded mRNA Sample Preparation Kit.  Briefly, 500 ng of total RNA was purified by oligo-dT beads selecting for polyadenylated RNA species and fragmented by divalent cations under elevated temperature. The fragmented RNA underwent first strand synthesis using reverse transcriptase and random primers.  Second strand synthesis created the cDNA fragments using DNA polymerase I. Following RNaseH treatment, the cDNA fragments went through end repair, adenylation of the 3’ ends, and ligation of adapters.  The cDNA library was enriched using 8 cycles of PCR and purified. Quality control consisted of assaying the final library size using the Agilent Bioanalyzer and quantifying the final library by RT- PCR and PicoGreen (fluorescence) methods. A single peak between 250-350 bp indicated a properly constructed and amplified library ready for sequencing.