GSM1406253: RM-Bud2; Solanum lycopersicum; RNA-Seq
Total RNA was isolated from 1-2 mm floral buds at stage 6-8 of three individual 35S::REVRis (T2 generation of Line 1) plants and corresponding WT controls by TRIZOL method. RNA quantity and quality were assayed using the Agilent 2100 Bioanalyzer (Agilent Technologies). Three aliquots of RNA from transgenic or WT plants were pooled, respectively, and sent to Illumina Cluster Station and Illumina HiSeq™ 2000 System (BGI Inc.) for RNA library construction and deep sequencing. Specified Experimental Process: Extract 6 μg total RNA, use Oligo(dT) magnetic beads adsorption to purify mRNA, and then use Oligo(dT) as primer to synthesize the first and second-strand cDNA. The 5' ends of tags can be generated by two types of Endonuclease: NlaIII or DpnII (see table 1 for recognition sites). Usually, 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 17bp 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, 105bp fragments are purified by 6% TBE PAGE Gel electrophoresis. After denaturation, the single-chain molecules are fixed onto the Illumina Sequencing Chip (flowcell). Each molecule grows into a single-molecule cluster sequencing template through Situ amplification. Then add in four types of nucleotides which are labeled by four colors, and perform sequencing with the method of sequencing by synthesis (SBS). Each tunnel will generate millions of raw reads with sequencing length of 49bp.