title: RNA-seq of mus musculus:embryo condyles Wt 2
design: The library construction starts with total RNA, which is enriched for mRNA with polyA tails using Oligo(dT) magnetic beads. The mRNA is then randomly fragmented using divalent cations in Fragmentation Buffer. This fragmented mRNA serves as the template for first-strand cDNA synthesis using random oligonucleotides as primers in an M-MuLV reverse transcriptase system. Following this, the RNA strand is degraded with RNase H, and the second cDNA strand is synthesized using DNA polymerase I and dNTPs. The double-stranded cDNA is purified and subjected to end repair, A-tailing, and adapter ligation. cDNA fragments of approximately 370 to 420 bp are selected using AMPure XP beads, PCR-amplified, and the PCR products are purified again with AMPure XP beads to obtain the final library. After library construction, an initial quantification is performed using a Qubit 2.0 Fluorometer, diluting the library to 1.5 ng/l. The library's insert size is then analyzed with an Agilent 2100 bioanalyzer. Once the insert size meets expectations, the effective concentration of the library is accurately quantified by qRT-PCR (with an effective concentration above 2 nM) to ensure library quality.