GSM9896044: HACAT cells,treatment,1; Homo sapiens; RNA-Seq
Extract total RNA using Invitrogen TRIzol reagent. The concentration and purity of total RNA were determined using a NanoDrop spectrophotometer (Thermo Scientific NanoDrop one, Thermo Fisher Scientific, Waltham, MA, USA), while RNA integrity was assessed through either RNA-specific agarose gel electrophoresis or the Agilent Fragment Analyzer 5400 system (Agilent Fragment Analyzer 5400, Agilent Technologies Inc., California, USA). mRNA enrichment was performed using the NEBNext® Poly(A) mRNA Magnetic Isolation Module (New England Biolabs Inc., Ipswich, Massachusetts, USA) with Oligo(dT) magnetic beads. The enriched mRNA was then randomly fragmented using divalent cations (ion interruption method) with the NEBNext Ultra II RNA Library Prep Kit for Illumina (New England Biolabs Inc., Ipswich, Massachusetts, USA). Using the fragmented mRNA as a template, double-stranded cDNA was synthesized with random oligonucleotides as primers (for strandspecific library construction, dUTP replaced dTTP in the dNTP mix during second-strand synthesis). The double-stranded cDNA was purified, followed by end repair, A-tailing, and adapter ligation. cDNA fragments of approximately 400 – 500 bp were size-selected using AMPure XP beads (Beckman Coulter, Beverly, CA, USA), PCR-amplified, and the PCR products were purified again with AMPure XP beads to obtain the final library. Library fragment sizes were analyzed using an electrophoresis-based instrument such as the LabChip GX. Total library concentration was measured using Pico green, and the effective concentration of the library was quantified by qPCR.