GSM5229498: lo_EV rep2; Homo sapiens; RNA-Seq
Total RNA was isolated with a modified Trizol protocol. Libraries were prepared for RNA-Seq using KAPA Biosystems KAPA mRNA HyperPrep Kit, according to manufacturer's directions. Briefly, total RNA (0.1-1 ug) is enriched for polyadenylated sequences using oligo-dT magnetic bead capture. The enriched mRNA fraction is then fragmented, and first-strand cDNA is generated using random primers. Strand specificity is achieved during second-strand cDNA synthesis by replacing dTTP with dUTP, which quenches the second strand during amplification. The resulting cDNA is A-Tailed and ligated with indexed adapters. Finally, the library is amplified using a DNA Polymerase which cannot incorporate past dUTPs, effectively quenching the second strand during PCR. KAPAHyperPrepmRNA