RNA-Seq of murine syngeneic model
Sequencing libraries will be created using the IlluminaTruSeq Stranded mRNA method, which preferentially selects for messenger RNA by taking advantage of the polyadenylated tail.   Briefly, total RNA samples are concentration normalized, and poly-adenylated RNA is purified using oligo-dT attached to magnetic beads.  Purified mRNA is fragmented using heat in the presence of divalent cations. The fragmented RNA is converted into double-stranded cDNA, with dUTP utilized in place of dTTP in the second strand master mix.  This facilitates the preservation of strand information, as amplification in the final PCR step will stall when it encounters Uracil in the nucleotide strand, rendering the first strand as the only viable amplification template.   The double stranded cDNA undergoes end-repair, A-tailing, and ligation of adapters that include index sequences.  The final libraries are amplified via polymerase chain reaction (PCR), after which they are quantified, normalized and pooled in preparation for sequencing.