CTAB-method library construction, and sequencing were carried out at Beijing Genome Institute at Shenzhen, China. An extract of 8 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 can be generated by two types of endonuclease: NlaIII or DpnII. Then, the bead-bound cDNA is subsequently digested with restriction enzyme NlaIII, which recognizes and severs the CATG sites. Fragments other than 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 the Illumina adaptor 1 and the CATG site constitutes the recognition site of MmeI, which is a type of endonuclease with separate recognition and digestion sites; it breaks 17 bp downstream of the CATG site, producing tags with adaptor 1. After removal of 3′ fragments with magnetic beads precipitation, the Illumina adaptor 2 is ligated to the 3′-ends of tags, acquiring tags with different adaptors of both ends to form a tag library.
GEO Accession
GSM2949436