P-GSE39151-3 - P-GSE39151-3
nucleic acid library construction protocol
Total RNA of T. spiralis (Ad, NBL and ML) was purified using Trizol reagent. 6 μg of total RNA from each preparation was treated with Oligo-(dT) conjugated magnetic beads to purify mRNA. Double-strand cDNA was synthesized guided by the Oligo-(dT) as a primer and then digested with the endonuclease NlaⅢ that recognizes the CATG sites. The Illumina adaptor 1, containing a MmeI restriction site, was added to the cDNAs attached to the magnetic beads, which was further digested with MmeI. Following MmeI digestion and dephosphorylation, cDNA fragments were purified and the Illumina adaptor 2 was ligated to the 3’ends of the tags to create tag library with different adapters at both ends. After15 cycles of linear PCR amplification, 95 bp fragments were purified from 6% TBE PAGE gels and attached to the Illumina sequencing chip for sequencing.