GSM9197216: treatment-group1; Mus musculus; RNA-Seq
RNA is extracted from tissues or cells using standard extraction methods, followed by rigorous quality control of the RNA samples. The quality control primarily involves quantification using a Nanodrop and detection of RNA integrity using an Agilent 4200 TapeStation Utilizing the structural characteristic that most eukaryotic mRNA carries a polyA tail, Oligo(dT) magnetic beads are used to enrich mRNA with a polyA tail. Subsequently, the obtained mRNA is randomly fragmented using divalent cations. Using the fragmented mRNA as a template and random oligonucleotides as primers, the first strand of cDNA is synthesized in the 1st Strand Enzyme reverse transcriptase system. Then, the second strand of cDNA is synthesized using 2nd Strand Enzyme and dNTPs. The purified double-stranded cDNA undergoes end repair, A-tail addition, and ligation of sequencing adapters. cDNA fragments of approximately 200-300 bp are selected, PCR amplified, and the PCR products are purified again to finally obtain the library. Afterwards, the library is quantified using a Qubit 2.0 Fluorometer and diluted to 1.5 ng/ul. The insert size of the library is detected using an Agilent 4200 TapeStation to ensure library quality. Finally, Illumina sequencing is performed on the qualified library after library inspection.