GSM8896731: OVU2AF2sinc3; Homo sapiens; RNA-Seq
Total RNA was extracted from cell samples, and a total of 1 μg of RNA was required for mRNA sequencing library construction. 1.mRNA Enrichment  For eukaryotic mRNA, enrichment was performed using Oligo (dT) magnetic beads to isolate poly(A)-tailed mRNA.  2.mRNA Fragmentation  The enriched mRNA was randomly fragmented into short fragments of approximately 200 bp using chemical or enzymatic methods.  3.Strand-Specific cDNA Synthesis  First-strand cDNA was synthesized using fragmented mRNA as a template and random primers for reverse transcription.  During second-strand cDNA synthesis, dTTP in the dNTP mix was replaced with dUTP to enable strand specificity.  4.Adapter Ligation  Double-stranded cDNA was purified using AMPure XP beads.  The cDNA ends were repaired and A-tailed using an End Repair Mix.  Sequencing adapters were ligated to the A-tailed cDNA.  5.Second-Strand cDNA Digestion  USER enzyme was used to digest the second-strand cDNA (containing dUTP), ensuring the final library contained only first-strand cDNA.  6.PCR Enrichment  The cDNA library was amplified by PCR for approximately 15 cycles using primers complementary to the sequencing adapters to enrich the final library.  7.Library Quality Control and Quantification  The quality of the library was assessed using an Agilent Bioanalyzer or similar instrument to confirm the expected fragment size distribution (~200-500 bp).  The library concentration was quantified using qPCR or a fluorometric method (e.g., Qubit) to ensure optimal concentration for sequencing.