GSM8230314: HepG2 cells, wild type, ONT DRS; Homo sapiens; RNA-Seq
Total RNAs were extracted from HepG2 cells using the standard TRIzol (Invitrogen) protocol. Briefly, cells were homogenized in 1 ml of TRIzol reagent and incubated at 4°C for 10 minutes. Next, the total RNA precipitates were collected as pellets after appropriate centrifugation. RNA pellets were then washed with 75% ethanol and resuspended in RNase-free water for further use. To purify poly-adenylated RNAs, the kit NEBNext® Poly(A) mRNA Magnetic Isolation Module (NEB #E7490S/L) was used. All steps were performed according to the manufacturer's instructions to isolate intact poly(A)+ RNAs with Oligo dT Beads d(T)25. Last, concentrations and purity were measured by the NanoDrop ND1000 Spectrophotometer and the Qubit Fluorometer to ensure the quality of harvested RNAs. 500 ng of poly(A)+ RNAs from HepG2 samples were used to perform Direct RNA sequencing on the PromethION sequencers (Oxford Nanopore Technologies Ltd., ONT). All RNA libraries were prepared using the RNA-SQK002 Kit and sequenced on R9.4.1 flowcells, following the guidelines provided by ONT.