GSM7717347: HepG2 siIGF2BP2 0h rep1; Homo sapiens; RNA-Seq
Generally, to isolate total RNA from cells, the media was aspirated, and the cells were washed once with a proper volume of ice-cold DPBS buffer for each plate. Then total RNA was isolated from cells with TRIzol reagent (Invitrogen) and then extracted following the manufacturer's protocol through isopropanol precipitation. GlycoBlue Coprecipitant (15 mg ml-1, Thermo Fisher Scientific) was added to the solution during precipitation if needed. Sample collection: five 10-cm plates of HepG2 cells were transfected with IGF2BP1-3 siRNA or METTL1 siRNA or control siRNA at 30% confluency. After 6 h, each 10-cm plate was re-seeded into three 6-cm plates, and each plate was controlled to afford the close numbers of cells. After 48 h, actinomycin D was added to 5 μg ml–1 at 6 h, 3 h, and 0 h before collection. The total RNA was purified with TRIzol (Invitrogen) described before. ERCC RNA spike-in control (Ambion) was added to each sample (0.02 µl per 1 µg total RNA). The total RNA with spike-in controls were then purified to acquire mRNA. Libraries were constructed with SMARTer Stranded Total RNA-Seq Kit v2 (Takara) according to the manufacturer's protocols. All libraries were sequenced on Illumina Nova Seq with single-end 100 bp read length.