title: method for vp sample experiment
design: The 3'-tag digital gene expression libraries were prepared using Illumina Gene Expression Sample Preparation Kit according to the manufacturer's instructions. Briefly, 6µg total RNA was used for mRNA capture with magnetic oligo(dT) beads. The first and second cDNA strands were synthesized, and the stranded bead-bound cDNA was subsequently digested with NlaIII. The 3'-cDNA fragments attached to the oligo(dT) beads were ligated to the Illumina Adapter 1, which contained a recognition site for the endonuclease MmeI for cutting 17 bp downstream of the recognition site (CATG) to produce tags with adapter 1. After removing 3' fragments with magnetic beads precipitation, an Illumina GEX adapter 2 was introduced at the site of MmeI cleavage. The resulting adapter-ligated cDNA tags were amplified using PCR-primers that were annealed to the adaptor ends for 15 cycles. The 85 base fragments were purified and recovered by 6% polyacrylamide Tris– borate–EDTA gel. The final quality of tagged sequences was checked by Agilent 2100 Bioanalyzer.