GSM1207283: maize_DGE_I; Zea mays; RNA-Seq
All the samples were harvested and immediately frozen in liquid nitrogen and stored at -80°C. The total RNA from each sample was then isolated using Trizol (Invitrogen) according to the manufacturer’s instructions. For RNA library construction and deep sequencing, 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 ﬁrst- and second-strand cDNA. The 5' ends of tags were generated by two endonucleases Nla III or Dpn II. The bead-bound cDNA was subsequently digested with restriction enzyme Nla III, 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 Mme I, which is a type of Endonuclease with separates recognition and digestion sites. It cuts 17 bp 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. Raw data (tag sequences) were deposited in the GEO database (No.GSE43058).