post-PHx 0h control 2 (CRX870142)
After qualifying RNA samples, eukaryotic mRNA is enriched using magnetic beads coated with Oligo(dT). Subsequently, the mRNA is fragmented using a fragmentation buffer to create short fragments. Using the mRNA as a template, a single-stranded cDNA is synthesized with random hexamers, followed by the addition of buffer, dNTPs, DNA polymerase I, and RNase H to generate double-stranded cDNA. The purified double-stranded cDNA is then cleaned using AMPure XP beads. The purified double-stranded cDNA undergoes end repair, A-tailing, and sequencing adapter ligation, followed by fragment size selection using AMPure XP beads. Finally, PCR amplification is performed, and the PCR products are purified using AMPure XP beads to obtain the ultimate sequencing library.