Illumina sequencing of sweet potato root transcript paired-end library
Illumina Solexa sequencing using the GAII platform was performed according to the manufacturer's instructions (Illumina). Briefly, poly (A) RNA was isolated from 20µg total RNA using Sera-mag Magnetic Oligo (dT) Beads (Illumina). To avoid priming bias when synthesizing cDNA, the purified mRNA was first fragmented into small pieces(100-400bp)using divalent cations at 94&deg;C for exactly 5 minutes. Then the double-stranded cDNA was synthesized using the SuperScript Double-Stranded cDNA Synthesis kit (Invitrogen) with random hexamer (N6) primers (Illumina). Following clean-up, the synthesized cDNA was subjected to end-repair and phosphorylation using T4 DNA polymerase, Klenow DNA polymerase and T4 PNK. These repaired cDNA fragments were 3'-adenylated using Klenow Exo- (3' to 5' exo minus,Illumina). Illumina Paired-end adapters were ligated to the ends of these 3'-adenylated cDNA fragments, followed by purification using MinElute PCR Purification Kit (Qiagen). To select a size range of templates for downstream enrichment, the products of the ligation reaction were purified on a 2% TAE-agarose gel (Certified Low-Range Ultra Agarose, Biorad). A range of cDNA fragments (200+/-25bp) was excised from the gel. Fifteen rounds of PCR amplification were performed to enrich the purified cDNA template using PCR Primer PE 1.0 and PCR Primer PE 2.0 (Illumina)] with Phusion DNA Polymerase.