Subcellular RNA Fractionation and RT-qPCR

To determine the ratio of nuclear to cytosolic mRNA for specific genes, subcellular RNA fractionation was carried out using an RNA Subcellular Isolation Kit (Active Motif, USA) according to the manufacturer's instructions. The reverse transcription PCR and the real-time quantitative PCR (RT-qPCR) were performed according to a modified version of the method described previously (28 (link)). Briefly, cDNA was synthesized using AMV Reverse Transcriptase (Takara Bio, Japan), and RT-qPCR was performed with a FastStart Essential DNA Green Master Kit (Roche Diagnostics, USA) using a Real-time PCR LightCycler 96 (Roche Diagnostics, Switzerland). The MIQE (29 (link)) checklist is provided in Supplementary Table S2.