GSM8689862: C57BL6 mice, rAAV8-Hnf1aos1, Bio3; Mus musculus; RNA-Seq
RNA was extracted using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's protocol. The first step involves ribosomal RNA (rRNA) depletion, where rRNA is removed to enrich for non-rRNA molecules such as mRNA, ensuring that the library focuses on the RNA of interest. Next, the RNA is subjected to fragmentation and random priming, breaking the RNA into smaller pieces to facilitate even sequencing coverage and initiating complementary DNA (cDNA) synthesis with random primers. Subsequently, the fragmented RNA is converted into double-stranded cDNA through first and second-strand cDNA synthesis. To prepare the cDNA for adapter ligation, the ends of the cDNA fragments undergo end repair, phosphorylation, and dA-tailing. This step ensures that the cDNA fragments have uniform and compatible ends for ligation with sequencing adapters. Finally, adapter ligation is performed, attaching specific adapters to the cDNA fragments for their identification and amplification. The library is then enriched through a PCR step to increase the quantity of adapter-ligated fragments. The final library is ready for high-throughput sequencing, enabling comprehensive RNA analysis.