DR4DQ8_31
The total RNA isolated as described above was used for 50 bp single-end RNA-Sequencing at the University of Minnesota Genomics Center (UMGC) on the Illumina HiSeq 2000. RNA quality was assessed with the Agilent Bioanalyzer, and samples with high RNA integrity number (RIN >8) were used for library construction following the manufacturers (Illumina) instructions. In summary, 1 microgram of total RNA was oligo-dT purified using oligo-dT coated magnetic beads, chemically fragmented and then reverse transcribed into cDNA. The cDNA was fragmented, blunt-ended, and ligated to indexed (barcoded) adaptors and amplified using 15 cycles of PCR. Final library size distribution was validated using capillary electrophoresis and quantified using fluorimetry (PicoGreen) and via Q-PCR.  Indexed libraries were normalized, pooled and then size selected to 320bp +/- 5% using Calipers XT instrument. TruSeq libraries were hybridized to a single end flow cell and individual fragments clonally amplified by bridge amplification on the Illumina cBot.  Once clustering was complete, the flow cell was loaded on the HiSeq 2000 and sequenced using Illuminas SBS chemistry. Approximately 50 milliion reads were sequenced for each sample. Base call (.bcl) files for each cycle of sequencing were generated by Illumina Real Time Analysis (RTA) software.  The base call files and run folders were then exported to servers maintained at the Minnesota Supercomputing Institute.  Primary analysis and de-multiplexing were performed using Illuminas CASAVA software 1.8.2.