GSM9437066: small intestine, model, rep2; Mus musculus; RNA-Seq
After rapid freezing with liquid nitrogen, the small intestine tissue was used to extract RNA using the Trizol method. Total RNA served as the starting material for RNA sample preparation, where mRNA was isolated using poly-T oligo-attached magnetic beads. Fragmentation was conducted under elevated temperature in the presence of divalent cations within the First Strand Synthesis Reaction Buffer. For first strand cDNA synthesis, random hexamer primers and M-MuLV Reverse Transcriptase were employed; this step was followed by second strand synthesis using DNA Polymerase I and RNase H. Subsequently, the overhanging ends of the cDNA were converted to blunt ends. After adenylation of the 3ʹ ends, adaptors with a hairpin loop structure were ligated to the cDNA molecules. The cDNA fragments were then size-selected to a target length range of 370–420 bp using the AMPure XP system. Following size selection, PCR amplification was performed with Phusion High-Fidelity DNA Polymerase and universal primers.