CK-2 (CRX1984163; CRA030168)
Total RNA is extracted from tissue samples. Eukaryotic mRNA features a polyA tail at its 3' end. By utilizing magnetic beads coated with Oligo(dT) to form A-T base pairing with the polyA sequence, mRNA can be isolated from total RNA for transcriptomic analysis. The enriched mRNA represents intact RNA sequences, which are randomly fragmented into small fragments of approximately 300 bp. Under the action of reverse transcriptase and using random primers, a single-stranded cDNA is synthesized using mRNA as a template. Subsequently, double-stranded synthesis occurs, forming a stable double-stranded structure. The double-stranded cDNA structure features sticky ends. An End Repair Mix is added to blunt these ends, followed by the addition of a single A base at the 3' end to facilitate subsequent adapter ligation. The adapter-ligated products undergo purification and fragment selection. PCR amplification is performed using the selected fragments, and the resulting products are purified to obtain the final library.