DGE analysis the Heterosis in Early Maize Ear Inflorescence Development-Mo17 SAU
Tag library preparation for the three genotypes (B73, Mo17 and their F1) was performed in parallel by using the Illumina gene expression sample preparation kit as described previously . An extract of 6 µg of total RNA was obtained and treated with Oligo(dT) magnetic bead adsorption to purify mRNA. Oligo(dT) was then used as a primer to synthesize the ?rst- and second-strand cDNA. The 5' ends of tags were generated by two endonucleases NlaIII or DpnII. The bead-bound cDNA was 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 were washed away and the Illumina adaptor 1 was 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 separate recognition and digestion sites. It cuts 17bp downstream of the CATG site, producing tags with adaptor 1. After removing 3' fragments with magnetic beads by precipitation, Illumina adaptor 2 was ligated to the 3' ends of tags, acquiring tags with different adaptors at both ends to form a tag library. After 15 cycles of linear PCR amplification, 95bp fragments were purified by 6% TBE PAGE Gel electrophoresis. After denaturation, the single-chain molecules were fixed onto the Illumina Sequencing flowcell. Each molecule grows into a single-molecule cluster sequencing template through in situ amplification. The four types of nucleotides were labeled by four colors, and added to perform sequencing by synthesis (SBS) . Each tunnel will generate millions of raw reads with sequencing length of 35bp.
Sichuan Agricultural University
Ear Inflorescence Mo17 SAU