GSM6645251: OCI-LY19S rep3; Homo sapiens; RNA-Seq
The trizol-isolated total RNA samples were first quantitated by Qubit RNA BR Assay (Thermo Scientific) and assessed for their quality by Agilent bioanalyzer run using RNA 6000 Nano Kit to determine the RIN scores for the samples. The RNA samples were then treated with the RNase-free, Turbo DNase I (Thermo Scientific), followed by poly (A) mRNA enrichment using NEBNext® Poly(A) mRNA Magnetic Isolation Module according to manufacturer's instructions. Subsequently, the RNA library is prepared using the NEBNext® Ultra™ II Directional RNA Library Prep Kit according to manufacturer's recommendation. All libraries underwent 8 cycles of PCR with the recommended conditions. NEBNext oligos from NEB were ligated to allow for multiplexing of samples. After library preparation, the samples were quantitated and run on the Bioanalyzer using DNA High Sensitivity DNA Analysis kit to determine library molarities. Libraries were pooled which was then diluted to a final concentration of 5 nM. Pooled and diluted library was sent for next-generation RNA-seq by NovogeneAIT Genomics (Singapore). Library was loaded onto the Nextseq 500 and sequencing was performed using a 75-cycle high output v2 kit. The gene expression level is calculated by the reads per kilobase/million mapped reads method. Each sample was prepared as three biological triplicates to ensure the reliability of RNA-seq data.