Axd/Axd2
Samples were processed using Illuminas TruSeq Stranded mRNA LT sample preparation kit (p/n RS-122-2101) according to manufacturers instructions.  Deviations from the protocol were as follows: 250ng total RNA was used as starting material.  Fragmentation was carried out for 10mins instead of 8mins, and 14 cycles of PCR were used. Briefly, mRNA was isolated from total RNA using Oligo dT beads to pull down Poly-Adenylated transcripts. The purified mRNA was fragmented using chemical fragmentation (heat and divalent metal cation) and primed with random hexamers.  Strand-specific first strand cDNA was generated using Reverse Transcriptase and Actinomycin D.  This allows for RNA dependent synthesis while preventing spurious DNA-dependent synthesis.  The second cDNA strand was synthesised using dUTP in place of dTTP, to mark the second strand. The resultant cDNA is then A-tailed at the 3 end to prevent self-ligation and adapter dimerisation. Full length TruSeq adaptors, containing a T overhang are ligated to the A-Tailed cDNA.  These adaptors contain sequences that allow the libraries to be uniquely identified by way of a 6bp Index sequence. Successfully ligated fragments were enricheded with 14 cycles of PCR. The polymerase is unable to read through uracil, so only the first strand is amplified, thus making the library strand specific.