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After the RNA samples pass the quality inspection, eukaryotic mRNA is enriched using magnetic beads with Oligo(dT) (for prokaryotes, rRNA is removed using a reagent kit to enrich mRNA). Subsequently, the mRNA is fragmented using a fragmentation buffer, and single-stranded cDNA is synthesized using random hexamers as primers with mRNA as a template. The single-stranded cDNA is then converted into double-stranded cDNA using a buffer, dNTPs, DNA polymerase I, and RNase H. The purified double-stranded cDNA is then subjected to size selection using AMPure XP beads. Afterward, the cDNA undergoes end repair, A-tailing, and 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 final library. After library construction, initial quantification is performed using Qubit 2.0, followed by library fragment size detection using Agilent 2100. Once the inserted fragments meet the expected criteria, quantitative PCR (Q-PCR) is used to determine the effective concentration of the library. |
RNA-Seq |
TRANSCRIPTOMIC |
cDNA |
PAIRED
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