GSM9431121: MKN-45, NNMT knockdown 1; Homo sapiens; RNA-Seq
Total RNA was extracted using TRIzol reagent (15596018CN, Invitrogen, USA) following the manufacturer's protocol. Briefly, cells were lysed directly in culture dishes, chloroform was added for phase separation, and RNA was precipitated with isopropanol. The RNA pellet was washed with 75% ethanol and resuspended in RNase-free water. RNA concentration and purity were assessed using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA), and RNA integrity was evaluated by agarose gel electrophoresis or Agilent Bioanalyzer. RNA-seq libraries were prepared using a standard Illumina stranded mRNA library preparation protocol. Briefly, poly(A)+ RNA was enriched from total RNA using oligo(dT) magnetic beads, followed by fragmentation, first-strand and second-strand cDNA synthesis. After end repair, A-tailing, and adapter ligation, libraries were amplified by PCR and purified. Library quality and size distribution were assessed using an Agilent Bioanalyzer, and libraries were quantified prior to sequencing.