GSM8898612: HaCaT cells treated with AGE; Homo sapiens; RNA-Seq
Total RNA was isolated from the each sample using the RNeasy Mini Kit (Qiagen). Polyadenylated mRNA was purified using oligo dT beads included in the NEBNext Poly (A) mRNA Magnet Isolation Module (New England Biolabs, NEB). Purified mRNA was used for the construction of sequencing libraries using NEBNext Ultra II RNA library Prep kit (NEB) and NEBNext Multiplex Oligos for Illumina (NEB). The purified mRNA was incubated with NEBNext Random Primers in NEBNext First Strand Synthesis Reaction Buffer at 94°C for 15 min, and reverse transcription was performed with NEBNext Strand Synthesis Enzyme Mix. After reverse transcription, index sequences included in NEBNext Multiplex Oligos for Illumina were inserted to cDNA fragments with PCR amplification. Following purification with AMPureXP beads (Beckman Coulter), the quality and the concentration of the libraries was evaluated using a Tapestation 4200 D1000 (Agilent). The confirmed libraries were mixed to equal molecular amounts against the clustering and sequencing on an Illumina NovaSeq6000 DNA sequencer with 50 bp paired-end cycle sequencing kit (Illumina).