Sample preparation and sequencing on the Genome Analyzer (Illumina, San Diego, CA) were carried out according to the Illumina protocols with some modifications. Briefly, the double-stranded cDNA was treated with T4 DNA polymerase and the Klenow fragment for end repair. The 5′ end of the DNA fragments were then phosphorylated by T4 polynucleotide kinase, and an adenosine base was added to the 3′ end of the fragments by Klenow (3′-5′ exo−). A universal adaptor was then added to both ends of the DNA fragments by A-T ligation. Following 18 cycles of PCR with the Phusion DNA polymerase, the DNA library was then purified on a 2% agarose gel and fragments of 170–300 base-pair in size were recovered. Around 15 ng of the DNA library was then used for cluster generation on a grafted GAII Flow Cell, and sequenced on the Genome Analyzer for 36 cycles using the “Sequencing-by-synthesis” method.
