GSM9077229: WT-3-SAM; Oryza sativa; RNA-Seq
Total RNA was isolated by TRIzol reagent (Invitrogen) and purified by RNeasy Plant Mini Kit (Qiagen) After enriching eukaryotic mRNA with polyA tails using magnetic beads with Oligo(dT), the mRNA was fragmented with buffer. Using the fragmented mRNA as a template and random oligonucleotides as primers, the first strand of cDNA was synthesized in an M-MuLV reverse transcriptase system. Subsequently, the RNA strand was degraded with RNaseH, and the second strand of cDNA was synthesized in a DNA polymerase I system using dNTPs as raw materials. The purified double-stranded cDNA was subjected to end repair, addition of an A tail, and ligation of sequencing adapters. AMPure XP beads were used to select cDNA of approximately 200bp, followed by PCR amplification and purification of the PCR products with AMPure XP beads. Finally, the library was obtained.