GSM9438502: GastricMucosa_DIO_biol rep_1; Mus musculus; RNA-Seq
Total RNA was extracted using RNA STAT-60 (AMSBio). After chloroform extraction and phase separation, the aqueous phase was mixed with ethanol and loaded onto RNeasy Mini Kit columns (Qiagen) for purification and washing following the manufacturer's protocol. Sequencing libraries were generated using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following the manufacturer's recommendations. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature. First strand cDNA was synthesized using random hexamer primers and M-MuLV Reverse Transcriptase. Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends, adenylated, and ligated with NEBNext Adaptors. The library fragments were purified with the AMPure XP system and PCR amplified. Finally, the libraries were sequenced on an Illumina NovaSeq X Plus platform to generate 150 bp paired-end reads