title: Viral Sequencing Reads: Ad6 infected A549 6 hours replicate 1
design: mRNA Library Construction.  TruSeq mRNA libraries (Illumina, San Diego, CA) were generated for three treatments (mock, Ad6, Ad26) at two time points (6 and 12 hours).  RNA libraries were prepared according to the manufacturer’s instructions for the TruSeq RNA Sample Prep Kit v2 using an Eppendorf EpMotion 5075 robot (Hamburg, Germany).  Reverse transcription and adaptor ligation steps were performed manually.  Briefly, polyA mRNA was purified from total RNA using oligo dT magnetic beads. The purified mRNA was fragmented at 95°C for 8 minutes, eluted from the beads, and primed for first strand cDNA synthesis.  RNA fragments were reverse transcribed into cDNA using SuperScript III reverse transcriptase with random primers (Invitrogen, Carlsbad, CA).  Second strand cDNA synthesis was performed using DNA polymerase I and RNase H.  Double-stranded cDNA was purified using a single AMPure XP bead (Agencourt, Danvers, MA) clean-up step.  cDNA ends were repaired and phosphorylated using Klenow, T4 polymerase, and T4 polynucleotide kinase followed by a single AMPure XP bead clean-up.  A single 3’ adenosine was added to these blunt-ended cDNAs with Klenow exo-.  Paired-end DNA adaptors (Illumina) with a single “T” base overhang at the 3’ end were immediately ligated to the ‘A tailed’ cDNA population.  Unique indexes included in the standard TruSeq Kits (12-Set A and 12-Set B) were incorporated at the adaptor ligation step for multiplex sample loading on the flow cells.  The adapter-modified DNA fragments were purified by two rounds of AMPure XP bead clean-up steps and were enriched by 12 cycles of PCR using primers included in the Illumina Sample Prep Kit.  The concentration and size distribution of the libraries were determined on an Agilent Bioanalyzer DNA 1000 chip (Santa Clara, CA).  A final quantification, using Qubit fluorometry (Invitrogen, Carlsbad, CA), was done to confirm sample concentration.      mRNA Library Sequencing.  Libraries were loaded onto paired end flow cells at concentrations of 8-10 pM to generate cluster densities of 700,000/mm2 following Illumina’s standard protocol using the Illumina cBot and cBot Paired end cluster kit version 3.  Libraries were indexed on the flow cell accommodating 3 or 4 libraries per lane.  The flow cells were sequenced as 101 X 2 paired end reads on an Illumina HiSeq 2000 using TruSeq SBS sequencing kit version 3 and HCS v2.0.12 data collection software.  Base-calling was performed using Illumina’s RTA version 1.17.21.3.  An initial sequencing run was performed on single mock, Ad6, and Ad26 samples followed by a second batch of samples for a final "n" .