Illumina DGE results of Taxus mairei
The main reagents and supplies are Illumina Gene Expression Sample Prep Kit and Illumina Sequencing Chip (flowcell), and the main instruments are Illumina Cluster Station and Illumina HiSeq™ 2000 System. Six micrograms total RNA were extracted, oligo(dT) magnetic beads adsorption was used to purify mRNA, and then Oligo(dT) was used as primer to synthesize the first and second-strand cDNA. The bead-bound cDNA is subsequently digested with restriction enzyme NlaIII, which recognizes and cuts off the CATG sites. The fragments apart from the 3' cDNA fragments connected to Oligo(dT) beads are washed away and the Illumina adaptor 1 is ligated to the sticky 5' end of the digested bead-bound cDNA fragments. The junction of Illumina adaptor 1 and CATG site is the recognition site of MmeI, which is a type of endonuclease with separated recognition sites and digestion sites. It cuts at 17 bp downstream of the CATG site, producing tags with adaptor 1. After removing 3' fragments with magnetic beads precipitation, Illumina adaptor 2 is ligated to the 3' ends of tags, acquiring tags with different adaptors of both ends to form a tag library. After 15 cycles of linear PCR amplification, 95 bp fragments are purified by 6% TBE PAGE gel electrophoresis. After denaturation, the single-chain molecules are fixed onto the Illumina sequencing chip (flow cell). Each molecule grows into a single-molecule cluster sequencing template through in situ amplification. Four types of nucleotides which are labeled by four colors were then added, and sequencing by synthesis (SBS) was performed. Each tunnel will generate millions of raw reads with sequencing length of 35 bp.