PolyA RNA Extraction from Total RNA

PolyA RNA was extracted from total RNA by oligo-dT affinity chromatography (NucleoTrap mRNA Kit, Macherey-Nagel, Allentown, PA, USA) following the instructions [34 (link),35 (link)]. Briefly, 130 ug of total RNA was mixed with oligo-dT latex beads and incubated at 68 °C for 5 min, then at room temperature for 12 min. After being centrifugated at 2000× g and 11,000× g, the pallets were washed three times on the microfilter and dried by centrifugation at 11,000× g for 1 min. At last, polyA RNA was incubated with DEPC-ddH₂O for 7 min at 68 °C, then centrifugated for 1 min at 11,000× g. The polyA RNA was purified using spectrophotometry (NanoDrop, Thermo Scientific, Waltham, MA, USA).