GSM8289912
Total RNA from the HUH7 and PLC/PRF/5 was extracted using Trizol reagent (Thermo Scientific, MA, USA). The extracted RNA was fragmented using Fragmentation Buffer. m6A Antibody (in kit) was then added, followed by incubation for 4 hours. Enrichment of mRNA was performed using Oligo d(T) magnetic beads. The mRNA was fragmented into approximately 100bp short fragments using a divalent cation. Immunoprecipitation was carried out using an m6A antibody. The RNA obtained from IP was used as a template to synthesize single-stranded cDNA using a hexamer random primer. Splint adapters were ligated and the resulting products were purified using magnetic beads and amplified by PCR. The constructed library was quality-checked using agarose gel electrophoresis. The library was quantified using Qubit 3.0 to determine if the library concentration was suitable for sequencing. Once the library passed quality control, different libraries were sequenced on the machine according to the required effective concentration and target data output.