GSM9533659: Akata EBV+ sgNC; Homo sapiens; RNA-Seq
Total RNA was isolated using the ISOLATE II RNA Mini Kit (Merdian Bioscience) according to the manufacturer's instructions. Briefly, cell pellets containing up to 5 × 106 cells were lysed in RLY buffer supplemented with β-mercaptoethanol and homogenized by vortexing. Lysates were clarified using ISOLATE II filters, and RNA binding conditions were adjusted by the addition of 70% ethanol. Samples were loaded onto silica spin columns, followed by membrane desalting and on-column DNase I treatment to remove genomic DNA. Columns were sequentially washed with RW1 and RW2 buffers, dried by centrifugation, and total RNA was eluted in RNase-free water. For gene expression analysis by qPCR, the cDNA synthesis was performed using High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Cat. #4374966, Applied Biosystem). RNA-seq libraries were prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina according to the manufacturer's instructions (NEB, Ipswich, MA, USA). Briefly, polyadenylated mRNA was enriched using oligo(dT) beads and subsequently fragmented for 15 minutes at 94 °C. First- and second-strand cDNA synthesis was performed, followed by end repair and 3′ adenylation. Universal adapters were ligated to the cDNA fragments, and indexed libraries were generated by limited-cycle PCR amplification. Library quality was evaluated using the Agilent TapeStation, and library concentration was determined using a Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA) and qPCR (KAPA Biosystems, Wilmington, MA, USA). Sequencing libraries were clustered on a flow cell and sequenced on an Illumina platform (HiSeq 4000 or equivalent) following the manufacturer's protocols